Use of antibodies against O-acetylated GD2 gangliosides to improve the therapeutic potential of drugs

Abstract

A novel composition and kit are provided that overcome the drawbacks of drug uptake in cancer cells and tumors, improve their therapeutic potential, and also suppress their side effects, such as toxicity. [Solution] A composition for use in delivering an anticancer drug into cells expressing OAcGD2 ganglioside, comprising: (i) at least one anticancer drug having a molecular weight of 100 to 200,000 daltons; and (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside, wherein the multimeric antibody comprises a light chain variable region (VL) polypeptide and a heavy chain variable region (VH), the multimeric antibody generates a non-selective pore in the cell membrane, and the multimeric antibody comprises an IgG1 constant region mutated to have multimerization properties.

Description

Technical Field

[0001] This patent application claims priority to European Patent Application Publication No. 16002576.3, filed on December 5, 2016, which is incorporated herein by reference.

[0002] The present invention relates to a composition comprising an anti-cancer agent and an antibody that recognizes a specific O-acetylated form of GD2 ganglioside (i.e., OAcGD2 ganglioside) that increases the uptake of the anti-cancer agent by cancer cells, and their use in a method of treating, preventing and / or managing a specific cancer, characterized by cells expressing OAcGD2 ganglioside.

Background Art

[0003] Delivery of anti-cancer agents to cells is a central aspect of treatment approaches for cancer. The biochemical properties of anti-cancer agents depend on their molecular weight, environmental pH and their lipophilicity versus hydrophilicity profile.

[0004] Passing through the negatively charged cell membrane is the first step in delivering anti-cancer agents to cells. It is now well established that a pH gradient occurs in animal tumors. Since anti-cancer agents are ionizable in an aqueous medium, the acidic extracellular pH in tumor cells differentially modulates the activity of weakly basic or acidic anti-cancer agents (Non-Patent Document 1). In solution, most anti-cancer agents exist as both non-ionized and ionized forms. Non-ionized anti-cancer agents are usually more lipophilic and can easily diffuse through the cell membrane. In contrast, ionized anti-cancer agents are less lipophilic and usually cannot permeate the lipid membrane without parasitizing the transporters used for physiological substrates. Thus, this ion-trapping mechanism can change drug accumulation and considerably regulate chemotherapy efficiency.

[0005] The ability to overcome the biological barriers that arise in delivering anticancer drugs to cancer cells and tumors must be balanced with toxicity. Among conventional anticancer drugs, alkylating agents, antimetabolites, or antitumor antibiotics do not directly damage or interfere with DNA or RNA, thereby preventing cancer cells from growing and proliferating. Other drugs, such as topoisomerase inhibitors or mitotic inhibitors, specifically target enzymes involved in the cell cycle. Different drugs or hormones are also used to mature cancer cells into normal cells or to slow cancer growth. Almost all anticancer drugs are toxic, and chemotherapy can produce serious, often dangerous side effects, including severe nausea, myelosuppression, and immunosuppression.

[0006] Furthermore, exposure to conventional anticancer drugs can lead to chemotherapy failure and tumor progression, even when administered in combination. This phenomenon is often due to pre-existing progenitor cells or newly formed drug-resistant clones. In addition, anticancer drugs can cause serious side effects such as cardiac or nerve damage, or, more dramatically, increase the risk of secondary cancers several years after drug administration.

[0007] More recently, RNA-targeted drugs in human cells have been developed by utilizing the ability of RNA interference to arrest the expression of target genes with high efficiency and specificity (Non-Patent Literature 2). RNA-based therapies developed for cancer treatment rely on the use of double-stranded synthetic short RNA molecules (miRNAs) or synthetic DNA / RNA-like oligonucleotides (ASOs). The movement of these molecules across the cell membrane of cancer cells is quite problematic due to their negative charge. This drawback can be mitigated by chemically modifying the oligonucleotide structure and / or by using delivery systems including viral vectors, biocompatible cationic polymers and copolymers, inorganic nanoparticles, atelocollagen, and liposomes. However, these delivery systems can affect the pharmacokinetic processes of miRNA-based therapies and may also be toxic. Furthermore, delivery systems often require parameter optimization for each cell type.

[0008] Therefore, there is a need for new compositions and methods that overcome the shortcomings of drug uptake in cancer cells and tumors, thereby improving their therapeutic potential and suppressing their side effects, such as toxicity. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] MAHONEY et al., Biochemical Pharmacology, vol.66, p:1207~1218, 2003 [Non-Patent Document 2] BARATA et al., Cancer Treatment Reviews, vol.50, p:35~47, 2016 [Overview of the project] [Means for solving the problem]

[0010] The inventors previously demonstrated that a mouse therapeutic antibody specifically targeting the O-acetylated form of GD2 ganglioside (i.e., OAcGD2 ganglioside) exhibits beneficial effects in treating cancers expressing OAcGD2 ganglioside (European Patent No. 2076542B1).

[0011] The inventors have now discovered, surprisingly, that this antibody, in addition to its own anticancer activity, is effective in treating cancer when used as an adjuvant to other anticancer agents. More specifically, they demonstrate a synergistic effect in combination therapy of an antibody specifically targeting OAcGD2 ganglioside with various anticancer agents against cancers expressing OAcGD2 ganglioside, making it possible to reduce the dosage of anticancer agents and, consequently, the toxic side effects.

[0012] Accordingly, the present invention relates to a composition for the delivery of an anticancer agent to cells expressing OAcGD2 ganglioside, comprising (i) at least one anticancer agent and (ii) at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside, wherein the at least one antibody that recognizes OAcGD2 ganglioside is advantageous to the uptake of the at least one anticancer agent by the cell and, consequently, to its activity. Herein, the at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside is specific to the ganglioside.

[0013] In one embodiment, at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is, a) Light chain variable region (VL) polypeptide of amino acid sequence SEQ ID NO: 1, and b) Heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 2 Includes.

[0014] Furthermore, in some embodiments, at least one anticancer agent is selected from the group comprising or consisting of anticancer agents, alkylating agents, antimetabolites, antitumor antibodies, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors, corticosteroids, hormones or hormone-like drugs, cytokines, nucleoside analogs, nucleic acids, double-stranded synthetic short RNA molecules (miRNAs), or synthetic DNA / RNA-like oligonucleotides (ASOs).

[0015] At least one anticancer drug may not be able to pass through the cell membrane of cancer cells on its own.

[0016] At least one of the anticancer drugs has a molecular weight of 100 daltons to 200,000 daltons.

[0017] Another object of the present invention is to provide an antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof, which can be used for selective delivery of anticancer agents into cells expressing OAcGD2 ganglioside in order to treat cancers expressing OAcGD2 ganglioside.

[0018] Another object of the present invention is to provide a method for delivering an anticancer agent into cells expressing OAcGD2 ganglioside, comprising the step of contacting cancer cells with at least one antibody that recognizes OAcGD2 ganglioside in an amount and concentration effective for increasing the uptake of the anticancer agent by the cells, wherein the antibody causes permeability defects such as pore formation in the cell membrane.

[0019] A further object of the present invention is to provide a method for preventing and / or treating cancer expressing OAcGD2 ganglioside, comprising the step of administering to a patient in need a composition comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0020] A further object of the present invention is to provide a method for enhancing sensitivity to anticancer agents in patients with cancer expressing OAcGD2 ganglioside, the method comprising the step of administering to a patient in need a composition comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0021] A further object of the present invention is to provide a method for preventing or delaying the development of anticancer drug-resistant cancer in patients with cancer expressing OAcGD2 ganglioside, the method comprising the step of administering to a patient in need a composition comprising (i) at least one anticancer drug, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0022] Another object of the present invention is to provide a method for identifying synergistic combinations of (i) at least one anticancer agent and (ii) at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside, which are suitable for the prevention and / or treatment of cancer expressing OAcGD2 ganglioside.

[0023] A further object of the present invention is to provide novel uses of antibodies that recognize OAcGD2 ganglioside, functional fragments thereof, or derivatives thereof, for increasing the intracellular uptake of anticancer drugs in cells expressing OAcGD2 ganglioside.

[0024] Finally, the present invention relates to a kit comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0025] [Figure 1A]This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 1B] This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 1C] This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 1D]This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 1E] This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 1F] This figure shows scanning electron microscopy results for neuroblastoma cell lines and glioblastoma primary cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. Untreated (or treated with isotype-matched negative control mouse IgG3) IMR5 (Panel A), LAN-1 (Panel C), and DUASOII (Panel E) cells share a continuous circle, while anti-OAcGD2mAb8B6-treated IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells exhibit several pores in their membranes. Magnifications are 2.5 kHz (KX) (Panels A, B, C, D, and F) and 2.0 kHz (Panel E), respectively. [Figure 2A]This figure shows the measurement of cell and pore diameter and surface area in neuroblastoma cell lines and glioblastoma primary cells: IMR5 (Panel A), LAN-1 (Panels B and C), and DUASOII (Panels D and E) cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. The magnifications are 5.02 KX (Panel A), 4.5 KX (Panels B and C), and 7.5 KX (Panels D and E), respectively. [Figure 2B] This figure shows the measurement of cell and pore diameter and surface area in neuroblastoma cell lines and glioblastoma primary cells: IMR5 (Panel A), LAN-1 (Panels B and C), and DUASOII (Panels D and E) cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. The magnifications are 5.02 KX (Panel A), 4.5 KX (Panels B and C), and 7.5 KX (Panels D and E), respectively. [Figure 2C] This figure shows the measurement of cell and pore diameter and surface area in neuroblastoma cell lines and glioblastoma primary cells: IMR5 (Panel A), LAN-1 (Panels B and C), and DUASOII (Panels D and E) cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. The magnifications are 5.02 KX (Panel A), 4.5 KX (Panels B and C), and 7.5 KX (Panels D and E), respectively. [Figure 2D] This figure shows the measurement of cell and pore diameter and surface area in neuroblastoma cell lines and glioblastoma primary cells: IMR5 (Panel A), LAN-1 (Panels B and C), and DUASOII (Panels D and E) cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. The magnifications are 5.02 KX (Panel A), 4.5 KX (Panels B and C), and 7.5 KX (Panels D and E), respectively. [Figure 2E]This figure shows the measurement of cell and pore diameter and surface area in neuroblastoma cell lines and glioblastoma primary cells: IMR5 (Panel A), LAN-1 (Panels B and C), and DUASOII (Panels D and E) cells incubated with 40 μg / ml anti-OAcGD2mAb8B6 for 30 minutes. The magnifications are 5.02 KX (Panel A), 4.5 KX (Panels B and C), and 7.5 KX (Panels D and E), respectively. [Figure 3] This figure shows the internal distribution of 5-FU, cisplatin, and doxorubicin in neuroblastoma cells. LAN-1 cells were treated with isotype-matched negative control mouse IgG3 (40 μg / ml) or combinations of anti-OAcGD2mAb8B6, anti-OAcGD2mAb c8B6.14b, and anti-OAcGD2mAb c8B6.15b (40 μg / ml) using either 5-FU (1.52 μM)-Panel A, cisplatin (1.52 μM)-Panel B, or doxorubicin (4.7 μM)-Panels C and D. The anticancer agents and mAbs were incubated at 37°C for 30 minutes. Neuroblastoma cells were fixed with 4% PFA before FACS acquisition. Positive controls were performed by treating fixed LAN-1 cells with 0.05% saponin combined with the anticancer agent for 30 minutes. The uptake of anticancer drugs in tumor cells is evaluated by flow cytometry (excitation with a 535 nm laser), and the results are expressed as the percentage of cells in the total cell population that have taken up the anticancer drug. [Figure 4-1]This figure shows the internal distribution of doxorubicin in melanoma (M21 cells), breast cancer (MDA-MB-231 cells), small cell lung cancer (H524 cells), and Ewing sarcoma cells (TC71 cells). Cells were treated with 0.26 μM of anticancer agents (except MDA-MB-231, 0.18 μM) in combination with isotype-matched negative control mouse IgG3 (40 μg / ml) or anti-OAcGD2mAb8B6 (40 μg / ml). The anticancer agents and mAbs were incubated at 37°C for 30 minutes. Tumor cells were fixed with 4% PFA before FACS acquisition. A positive control was performed by treating fixed tumor cells with 0.05% saponin in combination with the anticancer agent for 30 minutes. The uptake of anticancer drugs in melanoma (M21) - Panel A, breast cancer (MDA-MB-231) - Panel B, small cell lung cancer (H524) - Panel C, and Ewing sarcoma (TC71) - Panel D cells was evaluated by flow cytometry (excitation with a 535 nm laser), and the results were expressed as the percentage of cells in the total cell population that took up the anticancer drug. [Figure 4-2] This figure shows the internal distribution of doxorubicin in glioblastoma cells (DuGan). Cells were treated with 0.26 μM of anticancer agents (excluding MDA-MB-231, 0.18 μM) in combination with isotype-matched negative control mouse IgG3 (40 μg / ml) or anti-OAcGD2mAb8B6 (40 μg / ml). The anticancer agents and mAbs were incubated at 37°C for 30 minutes. Tumor cells were fixed with 4% PFA before FACS acquisition. A positive control was performed by treating fixed tumor cells with 0.05% saponin combined with the anticancer agent for 30 minutes. Anticancer agent uptake in glioblastoma (DuGan)-panel E cells was evaluated by flow cytometry (excited with a 535 nm laser), and the results are expressed as the percentage of cells that took up the anticancer agent in the total cell population. [Figure 5]This figure shows the enhancement of chemotherapy antitumor activity by anti-OAcGD2mAb8B6 in an in vivo mouse model of neuroblastoma experimental liver metastasis. Mice (n=10 / group) were inoculated with 2.5 × 10⁵ NXS2 mouse neuroblastoma cells by intravenous injection. 8B6mAb treatment was started on day 3 after tumor cell inoculation (cumulative dose = 150 μg, A-C) twice weekly for 3 consecutive weeks. All chemotherapy treatments were started on day 10 after tumor inoculation. Panel A: Isotretinoin was administered orally at 10 mg / kg five times a week for 2 weeks. Panel B: Topotecan was administered intraperitoneally at 0.36 mg / kg five times a week for 1 week. Panel C: Doxorubicin was administered intraperitoneally at 1 mg / kg five times a week for 2 weeks. Mice were euthanized on day 28 after tumor inoculation. Antitumor efficiency was evaluated by measuring the liver weight of the new sample compared to mice treated with vehicle alone. Liver weight indicates the number of liver metastases. The y-axis starts at 0.8g, which corresponds to the average normal liver weight. Data are shown as mean ± SEM. [Figure 6] This figure shows the systemic tolerance of anti-OAcGD2mAb8B6 in combination with isotretinoin (Panel A), topotecan (Panel B), and doxorubicin (Panel C). The mean weight of the mouse group (n=10) shown on day 0 in Figure 6 was defined as 100% weight. The weight of each group remained stable throughout the treatment period. △: Mice treated with mAb8B6; ○: Mice treated with anticancer drugs; □: Mice treated with chemotherapy drugs + mAb8B6. [Figure 7]This figure shows that anti-OAcGD2mAb8B6 overcomes tumor resistance in patient-derived TMZ-refractory GBM-10 cells (GBM-10TMZR) in an in vivo glioblastoma xenograft mouse model. 1 × 10⁶ GBM-10 cells were subcutaneously injected into mice on day 0. The mice were then divided into two groups. The first group of mice received a single dose of temozolomide (TMZ) at 0.05 mg / mouse by intraperitoneal injection on days 12, 22, and 32, while the second group of mice remained untreated. Tumor volume (Panel A) was monitored. OAcGD2 expression was evaluated in GBM-10 cells isolated from both TMZ-treated and untreated mice (Panel B). Next, the level of TMZ refractory status was determined by limiting dilution assays and expressed as the frequency of glioblastoma cancer stem cells (GSCs) in tumor xenografts. The effects of monotherapy (8B6 or TMZ) and a combination of the two drugs (TMZ + 8B6) on GSC survival were compared in GBM-10 cells isolated from untreated cells (Panel C, GBM-10 CTLs (TMZs)) and relapsed GBM-10 xenografts (Panel D, GBM-10 TMZR). [Modes for carrying out the invention]

[0026] In a first embodiment, the present invention relates to a method for delivering an anticancer agent into cells expressing OAcGD2 ganglioside, comprising the step of contacting cells with at least one antibody, a functional fragment or derivative thereof, that recognizes OAcGD2 ganglioside in an amount and concentration effective for increasing the uptake of the anticancer agent by the cells, wherein the antibody causes defects such as pore formation in the cell membrane.

[0027] Preferably, the at least one antibody that recognizes OAcGD2 ganglioside is a multimeric antibody.

[0028] In fact, the inventors have demonstrated that antibodies that recognize OAcGD2 ganglioside and simultaneously possess multimerizing properties (e.g., an agglutinating 8B6 IgG3 antibody, and a chimeric 8B6 antibody containing a chimeric IgG1 constant region that possesses multimerizing properties (i.e., hexamer formation)) induce the permeation of anticancer drugs, while antibodies that do not possess such multimerizing properties (e.g., a chimeric 8B6 antibody containing a chimeric IgG1 constant region) have no effect on such permeation. Therefore, it appears that both OAcGD2 ganglioside binding and multimerizing properties are necessary for pore formation in the cell membrane of cells expressing OAcGD2 ganglioside. In this specification, the term "multimeric antibody" may refer to a dimer, trimer, tetramer, ... or aggregate.

[0029] In this invention, the terms cell membrane, cytoplasmic membrane, outer cell membrane, and plasma membrane are synonymous and can be used without distinction.

[0030] The term "delivery of anticancer drugs into cells" refers to the release of anticancer drugs from within cells, which allows the drugs to reach their intracellular targets.

[0031] According to one preferred embodiment, cells expressing OAcGD2 ganglioside are tumor cells, cancer cells, cancer stem cells, or overproliferating cells having OAcGD2 ganglioside fixed to the cell membrane. The term “cancer stem cell” has its general meaning in the art and refers to a subpopulation of cancer cells that possess characteristics associated with normal stem cells, particularly the ability to generate all cell types found in a particular cancer sample (found in solid tumors and hematological malignancies). They possess the ability to self-replicate, differentiate into multiple cancer cell lineages, and proliferate extensively. They can induce new tumors with only a small number of cancer stem cells and tend to be resistant to conventional therapies, including chemotherapy and radiotherapy. Cancer stem cells have been identified in a wide variety of cancer types, including, but not limited to, leukemias, including acute myeloid leukemia and acute lymphoblastic leukemia; breast cancer; gliomas, including glioblastoma; colorectal cancer; pancreatic cancer; prostate cancer; lung cancer; liver cancer; bladder cancer; or gastric cancer. Various markers are observed when identifying cancer stem cells within a mass of cancer cells, and these markers are diverse and cancer type-dependent. Examples of markers that can be used to identify cancer stem cells include, but are not limited to, CD34, CD38, CD19, interleukin-3 receptor α (CD123), CD33, CD44, CD44v6, CD47, CD24, EpCAM (ESA), Lin, CD133, A2B5, SSEA-1, CD166, CD26, CD200, α2β1, Sca, CD45, Pecam, ALDH, ALDH1, Oct4, ABCG2, CXCR4, AFP, EMA, and IGF-IR.

[0032] The term "cellular uptake of anticancer drugs" refers to the contact and internal movement / permeation of anticancer drugs into target cells such as tumor cells, cancer cells, cancer stem cells, or overgrowth cells. It is now well established that crossing the cell membrane is the first rate-limiting step for anticancer drugs to reach their intracellular targets.

[0033] The term "defect" refers to a localized disruption of the cell membrane that results in a transient deformation of the cell that increases cell membrane permeability. For example, localized invagination or bulging of the cell membrane may result from the binding of at least one antibody that recognizes OAcGD2 ganglioside, its functional fragment, or a derivative thereof to cells expressing OAcGD2 ganglioside. In other words, a transient increase in cell membrane permeability of cells expressing OAcGD2 ganglioside occurs only in the presence of at least one antibody that recognizes OAcGD2 ganglioside, its functional fragment, or a derivative thereof. The defect may be transient, and therefore the cell membrane can return to its original structure.

[0034] The term "pore formation" refers to the physical modification or disturbance of the glycerophospholipid bilayer that forms holes within the cell membrane. This transiently increases the permeability of the cell membrane and can also play a role in the endocytosis of extracellular molecules. Pore formation allows molecules to enter the intracellular compartment from the extracellular compartment. The number and diameter of the pores depend on several parameters, including cell type, cell membrane composition, and the amount and duration of antibody contact with the cell. The pore size determines the size of the drug that can be delivered into the cytoplasm of a cell by the method of this invention. Pore size can be measured using scanning electron microscopy (SEM) and atomic force microscopy (AFM) (ZHAO et al., Journal of Drug Targeting, 16:1, 18-25, 2008).

[0035] According to one preferred embodiment, the average diameter of the film pores is about 1 nm to about 100 nm, preferably about 1 nm to about 50 nm, and most preferably about 1 nm to about 10 nm.

[0036] The lipid bilayer cell membrane is 4–10 nm thick. Therefore, the average diameter of the pores can, in some cases, be significantly larger than the cell membrane thickness.

[0037] In all embodiments, an antibody, functional fragment, or derivative thereof that recognizes OAcGD2 ganglioside generates nonselective pores on the cell membrane. “Nonselective pores” are intended to be pores that allow uncharged molecules or charged molecules, such as ionic anticancer agents, to diffuse through the phospholipid bilayer of the cell membrane. Pore size is a relevant criterion for the selection of uncharged or charged compounds that pass through the cell membrane.

[0038] When an antibody, functional fragment, or derivative that recognizes OAcGD2 ganglioside is brought into contact with cells expressing OAcGD2 ganglioside under appropriate conditions, it has the ability to induce cellular permeability of an anticancer drug into a given cell culture population, either partially or entirely, by creating pores within the cell membrane. "Cellular permeability" refers to the passage of an anticancer drug from the external environment into an intracellular environment under significantly better conditions than passive diffusion.

[0039] This discovery provides a completely new approach to treating cancers expressing OAcGD2 ganglioside. By acting as a nonselective pore in cancer cells, antibodies, functional fragments, or derivatives that recognize OAcGD2 ganglioside significantly increase the permeability of cancer cells to anticancer drugs. This can not only enhance their therapeutic effect but also reduce their associated potential side effects.

[0040] When used in vitro and / or ex vivo, the method of the present invention is performed for drug screening purposes. Therefore, the in vitro and / or ex vivo delivery method of anticancer agents into cells expressing OAcGD2 ganglioside, preferably tumor cells, is suitable for screening synergistic combinations of anticancer agents with antibodies that recognize OAcGD2 ganglioside, their functional fragments, or derivatives.

[0041] Accordingly, the present invention also relates to a composition for the delivery of an anticancer agent to cells expressing OAcGD2 ganglioside, preferably tumor cells, comprising (i) at least one anticancer agent, and (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof, wherein the at least one antibody that recognizes OAcGD2 ganglioside increases the uptake of the at least one anticancer agent by cells, preferably tumor cells.

[0042] When used in vivo, the method of the present invention is performed for therapeutic purposes. Therefore, the in vivo delivery method of an anticancer agent into cells expressing OAcGD2 ganglioside, preferably tumor cells, is suitable for the treatment of cancers expressing OAcGD2 ganglioside.

[0043] Accordingly, the present invention also relates to a composition used in a method for treating and / or preventing cancer expressing OAcGD2 ganglioside, comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0044] The present invention also relates to antibodies that recognize OAcGD2 ganglioside, functional fragments thereof, or derivatives thereof, used for the selective delivery of at least one anticancer agent into cells expressing OAcGD2 ganglioside for the treatment and / or prevention of cancer.

[0045] The present invention also relates to a method for preventing and / or treating cancer expressing OAcGD2 ganglioside, comprising the step of administering to a patient in need an effective amount of a composition comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0046] The terms "method for treating cancer expressing OAcGD2 ganglioside" or "method for treating cancer expressing OAcGD2 ganglioside" are synonymous and refer to curing, reversing, attenuating, mitigating, minimizing, suppressing or halting the adverse effects or progression of cancer expressing OAcGD2 ganglioside, or attenuating the progression of cancer expressing OAcGD2 ganglioside. Preferably, such treatment also results in a regression of tumor growth or metastatic spread, i.e., a reduction in the measurable size of the tumor. Most preferably, such treatment results in a complete regression of the tumor.

[0047] The term "OAcGD2 ganglioside-expressing cancer" refers to cancer having cells that express the O-acetylated form of GD2 ganglioside on their surface. Generally, such cells express more than 1,000 OAcGD2 ganglioside molecules on their cell surface, preferably more than 10,000, and more preferably more than 50,000. The OAcGD2 ganglioside-expressing cancers are selected from or include neuroblastoma, glioma, retinoblastoma, Ewing family of tumors, sarcomas (i.e., rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal cell carcinoma, head and neck cancer, and hematological cancers (i.e., leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and myeloma). More generally, the term “OAcGD2 ganglioside-expressing cancer” refers to cancer exhibiting OAcGD2 ganglioside-expressing cells in greater than 10%, preferably greater than 15%, and more preferably greater than 20%. Preferably, these cells are cancer stem cells (CSCs). Treatment of OAcGD2 ganglioside-expressing cancer refers to the administration of a composition for destroying tumors or cancer cells expressing OAcGD2 ganglioside on their surface, comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0048] "Prevention" of cancer expressing OAcGD2 ganglioside refers to the administration of a composition comprising (i) at least one anticancer agent, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier, for the purpose of reducing tumor growth, preventing metastasis and spread, or further preventing tumor recurrence. Preferably, the outcome of prevention may be the prevention of metastasis and spread in subjects already suffering from cancer expressing OAcGD2 ganglioside if treatment is administered before recurrence. Most preferably, another outcome of prevention may be the prevention of tumor recurrence in subjects who have already been treated for cancer expressing OAcGD2 ganglioside.

[0049] In this specification, the term “patient” refers to any mammal, including humans. The term is not limited to any particular age or sex. Therefore, adult and neonatal subjects, as well as children, are included in the term, regardless of whether they are male or female.

[0050] The term "anticancer agent" refers to a chemical, physical, or biological preparation or compound having antiproliferative, anticarcinogenic, and / or anticancer properties that can be used to inhibit tumor growth, proliferation, and / or development. Preferably, the anticancer agent has activity against cancers expressing OAcGD2 ganglioside.

[0051] At least one anticancer agent may be selected from the group including or comprising anticancer agents such as alkylating agents, antimetabolites, antitumor antibodies, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors, corticosteroids, hormones or hormone-like drugs, cytokines, nucleoside analogs, nucleic acids, such double-stranded synthetic short RNA molecules (miRNAs) or synthetic DNA / RNA-like oligonucleotides (ASOs).

[0052] At least one anticancer drug may not be able to pass through the cell membrane of cancer cells on its own.

[0053] In all embodiments, the anticancer agent has steric hindrance suitable for a pore size of approximately 1 nm to 2 μm and can pass through the cell membrane via a pore created by contact between the cell and an antibody that recognizes OAcGD2 ganglioside, its functional fragment, or a derivative thereof.

[0054] In some embodiments, anticancer agents can be embedded in biocompatible and biodegradable nanoparticles. This is particularly suitable when the anticancer agent is a nucleic acid, such as a double-stranded synthetic short RNA molecule (miRNA) or synthetic DNA / RNA-like oligonucleotide (ASO), or a short polypeptide that is protected from cleavage by a nuclease or protease.

[0055] In this case, the type of nanoparticle can be selected by those skilled in the art based on its size and its physicochemical and pharmacokinetic properties. For example, miRNA was successfully delivered to cancer cells using iron oxide nanoparticles in an animal model of gastric adenocarcinoma (HIRAKI et al., Molecular Therapy-Nucleic Acids (2015) 4, e231) and carbonate apatite nanoparticles in an animal model of colorectal adenocarcinoma (SUN et al., 2014, Mol Cell Biochem, vol.390, pp19~30). The size distribution of nanoparticles can be measured using various techniques such as atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). For example, the single particle size of iron oxide nanoparticles was 10~60 nm, those composed of carbonate apatite nanoparticles were 70~80 nm, and when aggregates were formed, they were up to 200~300 nm.

[0056] Nanoparticles can also be used in anticancer drugs used in chemotherapy, as they are uniformly distributed throughout the patient's body and make it impossible to distinguish cancer cells from normal cells.

[0057] In recent years, considerable effort has been focused on ultra-small inorganic particles (USNPs) for cancer treatment. Gold USNPs have a core size of 1-3 nm.

[0058] The size of the nanoparticles and micro-nanoparticles matches the size of the pores observed in the cell membrane of cancer cells expressing OAcGD2 ganglioside when incubated with an antibody that recognizes OAcGD2 ganglioside.

[0059] In some embodiments, the anticancer agent is not embedded in nanoparticles. In this case, the anticancer agent in solution is non-spherical, dynamic (rotating), and solvated. The apparent size of a dynamic, hydrated, and solvated anticancer agent can therefore be measured by utilizing the diffusivity of the anticancer agent. Dynamic light scattering (DLS) is considered the best technique for calculating the hydrodynamic radius (Rh), which is defined as the radius of an equivalent rigid sphere diffusing at the same velocity as the molecule being observed. Rh indicates the apparent size of the dynamically hydrated / solvated molecule. Rh is generally calculated from the diffusion coefficient using the Stokes-Einstein equation. The radius of gyration (Rg) is another parameter that allows for the measurement of molecular size, defined as the mass-weighted average distance from the core of the molecule to each mass element in the molecule. However, the Rg of a molecule can be obtained using other techniques such as small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), or from high-resolution X-ray structures.

[0060] It may also be appropriate to characterize the anticancer agent-forming portion of a composition used to treat cancer by its molecular weight.

[0061] In one preferred embodiment, the anticancer agent has a molecular weight of 100 daltons to 200,000 daltons.

[0062] "Molecular weight" means the average mass of the molecule calculated by summing the atomic weights of the atoms in the molecular formula that defines the molecule. The terms mass and weight are used interchangeably in the present invention. "Atomic weight" means the weighted average of the isotopic masses found in a typical terrestrial sample of the element as shown in the Mendeleev periodic table.

[0063] The definition of the dalton can be found in the Green Book (IUPAC, Green book, 3rd edition, "Quantities, Units and Symbols in physical chemistry", 2007). The dalton, symbol Da, is used as an alternative name for the unified atomic mass unit, symbol u. The dalton is related to the mass of the carbon-12 nuclide and is defined as 1 / 12 of the mass of a carbon-12 atom (m a ( 12 C) / 12 ≈ 1.666 538 782(83)×10 -27 kg = 10 -3 / Na(kg), where Na is Avogadro's number, 6.022 14×10 23 mol -1 (equal to). Thus, the molar mass of carbon-12 is exactly 12 g / mol. As a result, the molar mass is numerically identical to the atomic weight when expressed in SI units of g / mol (equal to g.mol -1 ). As an example, the molar mass of water (H2O) is 18 g.mol -1 and its molecular weight is 18 Da. The dalton can be combined with SI prefixes to express the mass of large molecules in kilodaltons (kDa) or megadaltons (MDa). For example, an anticancer agent with a molar mass of 200,000 g·mol -1 has a molecular weight of 200,000 Da and can also be defined as 200 kDa.

[0064] Also, in certain embodiments, the anticancer agent of the composition according to the present invention has a molecular weight of 10 kDa to 15 kDa, more preferably 11 kDa to 14 kDa, even more preferably 12 kDa to 13 kDa. Most preferably, the anticancer agent of the composition according to the present invention has a molecular weight of 10 kDa to 13 kDa.

[0065] In another embodiment, the anticancer agent of the composition according to the present invention has a molecular weight of 80 kDa to 200 kDa, more preferably 100 kDa to 180 kDa, and more preferably 120 kDa to 160 kDa. Most preferably, the anticancer agent of the composition according to the present invention has a molecular weight of 150 kDa to 200 kDa.

[0066] In another embodiment, the anticancer agent of the composition according to the present invention has a molecular weight of 120 to 800 daltons, more preferably 150 to 600 daltons, and more preferably 250 to 550 daltons. Most preferably, the anticancer agent of the composition according to the present invention has a molecular weight of 120 to 550 daltons.

[0067] Furthermore, in some embodiments, at least one anticancer agent is selected from the group of anticancer agents comprising or consisting of antitumor antibiotics such as anthracyclines, topoisomerase inhibitors such as camptothecin, alkylating agents such as imidazotetrazine or cyclophosphamide, prenolic lipids, antimetabolites such as diazines, and transition metal salts.

[0068] Anthracyclines are one of the most efficient classes of drugs used in the treatment of cancer. Beginning in the 1950s, these molecules were first identified from the soil bacterium Streptomyces peucetius. Anthracyclines consist of a rigid, planar tetracyclic structure with adjacent quinone and hydroquinone moieties, a short side chain with a carbonyl group at C-13, and the amino sugar daunosamine, which is linked to C-7 of the tetracyclic ring by a glycosidic bond. Anthracyclines enter cells by passive diffusion and can interact with topoisomerase-DNA complexes, thereby inhibiting cell proliferation. Despite their efficient activity in killing cancer cells, only a few of them, such as daunorubicin, doxorubicin, idarubicin, epirubicin, barurubicin, mitoxantrone, and picantrone, are approved for medicinal use. Furthermore, their clinical utility is limited by cumulative dose-dependent cardiotoxicity that can cause irreversible heart failure. Preferably, the anthracycline used in the composition of the present invention is selected from the group comprising or consisting of daunorubicin, doxorubicin, idarubicin, barurubicin and other derivatives thereof. Most preferably, the anthracycline used in the composition of the present invention is doxorubicin or one of its derivatives.

[0069] In all embodiments, the composition according to the present invention comprises a therapeutically effective amount of at least one anticancer agent and a therapeutically effective amount of at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof.

[0070] In this specification, the term “therapeutic dose” encompasses any amount that yields the desired therapeutic or biological effect. The therapeutic effect depends on the cancer expressing the OAcGD2 ganglioside being treated or the desired biological effect. Therefore, the therapeutic effect may be a reduction in the severity of symptoms associated with cancer expressing the OAcGD2 ganglioside, and / or (partial or complete) inhibition of the progression of cancer expressing the OAcGD2 ganglioside. The amount required to induce a therapeutic response can be determined based on the type of cancer, the patient’s age, health, physique, and sex.

[0071] According to one preferred embodiment, doxorubicin or one of its derivatives is administered at a dose of 40 mg / m² for adult subjects. 2 ~60mg / m 2 It can be administered at a dose of 1 to 3 days every 3 or 4 weeks. The maximum cumulative dose of one of the doxorubicin or its derivatives is 600 mg / m². 2 It should not exceed this amount. The dosage for children is 25 mg / m². 2 ~30mg / m 2 It is reduced to (MATTHAY et al., N Engl J Med, 1999, vol.341, pp.1165~1173). mg / m 2 To convert a unit dose to a mg / kg dose in humans, simply divide by 37 (NAIR and JACOB, J Basic Clin Pharm., 2016, vol.7(2):pp.27~31). The dosages described herein are for adult and / or child humans, but may be adjusted to suit the size of other mammals depending on their weight or square meter size.

[0072] Camptothecin is a heterocyclic compound comprising a planar pentacyclic ring structure containing a pyrrolo[3,4-beta]-quinoline moiety (rings A, B, and C), a conjugated pyridone moiety (ring D), and a single chiral center at position 20 within an alpha-hydroxylactone ring (ring E) in an (S) configuration. These quinoline alkaloids were first extracted from the Asian tree, *Camptothecin serrata*. Topotecan and irinotecan are the only two camptothecin derivative drugs whose use has been approved for the treatment of cancer. Preferably, the camptothecin used in the composition of the present invention is selected from the group comprising or consisting of topotecan, irinotecan, rubitecan, and other derivatives. Most preferably, the camptothecin used in the composition of the present invention is topotecan, irinotecan, or one of their derivatives.

[0073] According to one preferred embodiment, topotecan or one of its derivatives is administered to adults at an initial dose of 1.5 mg / m². 2 Then, every 5 days for 3 weeks, followed by a dose of 1 mg / m². 2 ~1.5 mg / m² 2 It can be administered for 5 days every 3 weeks. The dosage for children is 0.75 mg / m². 2 The frequency is reduced to every 5 days or 28 days (DI GIANNATALE et al., Phase II study of temozolomide in combination with topotecan (TOTEM) in relapsed or refractory neuroblastoma: a European Innovative Therapies for Children with Cancer-SIOP-European Neuroblastomastudy and Reza Rahbar, Carlos Rodriguez-Galindo, John G. Meara, Edward R. Smith, Antonio R. Perez-Atayde. 2 December 2013 Springer Science & Business Media).

[0074] According to another preferred embodiment, irinotecan or one of its derivatives is administered to adult subjects at a dose of 340-350 mg / m². 2 It can be administered every three weeks by intravenous infusion over 90 minutes (FRIEDMAN et al., J. Clin. Oncol., vol.27(28), pp:4733~4740, 2009).

[0075] The dosages described herein are for adult and / or child humans, but may be adjusted to suit the size of other mammals depending on their weight or square meter size.

[0076] Imidazolazine is a class of heterobicyclic compounds containing ortho-condensed imidazole and tetrazine rings. Of these compounds, temozolomide (Temodar®, Temodal®, TMZ) has been approved for the treatment of newly diagnosed glioblastoma multiforme (GBM) and also for refractory anaplastic astrocytoma. This prodrug needs to be activated to be active. Although clearly beneficial to some patients with glioblastoma, TMZ also induces progressive tumor growth associated with the emergence of TMZ resistance. Preferably, the imidazotetrazine used in the composition of the present invention is temozolomide or one of its derivatives.

[0077] According to one preferred embodiment, temozolomide or one of its derivatives is administered at a dose of 75 mg / m² for adult subjects. 2 ~200mg / m 2 This dose can be administered daily.

[0078] The dosages described herein are for humans, but can be adjusted to suit the size of other mammals and children, depending on weight or square meter size.

[0079] Prenolipids are synthesized primarily from five-carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate, which are produced via the mevalonate pathway. Simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the sequential addition of C5 units, making this classification convenient; the polyterpene subclass of these structures contains more than 40 carbon atoms (i.e., more than 8 isoprenoid units). For example, vitamin A and its derivatives, as well as phytanic acid and its oxidation product pristanic acid, are classified as C20 isoprenoids. Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A. Another biologically important class of molecules is exemplified by quinones and hydroquinones, which contain isoprenoid termini attached to a quinone-like core of non-isoprenoid origin. Vitamins E and K, as well as ubiquinone, are examples of this class. Among prenolipids, retinoids are natural and synthetic derivatives of vitamin A. Several studies have reported their use as potent differentiation inducers or as tumor cell apoptosis inducers for treating cancers such as acute myeloid leukemia (AML). Examples of vitamin A derivatives include retinoic acid (RA), all-trans retinoic acid (ATRA), 9-cisRA, 4-HPPR, 13-cisRA, and synthetic analogs of retinoic acid such as AM580. Recently, 13-cisRA has been found to be beneficial in the treatment of high-risk neuroblastoma after bone marrow transplantation (Matthay et al., N Engl J Med;341:1165~1173, 1999). Preferably, the prenolic lipid used in the composition of the present invention is selected from the group comprising or consisting of ATRA, 9-cisRA, and 13-cisRA. Most preferably, the prenolic lipid used in the composition of the present invention is 13-cisRA, also known as isotretinoin.

[0080] According to one preferred embodiment, isotretinoin is administered to adults at a dose of 80 mg / m². 2 ~160mg / m2 It can be administered as follows (YU et al., N Engl J Med., 2010, vol.363(14):pp.1324~1334).

[0081] The dosages described herein are for humans, but can be adjusted to suit the size of other mammals and children, depending on weight or square meter size.

[0082] Diazines are organic compounds comprising a five-membered heterocyclic compound having five nitrogen atoms and two nitrogen atoms at positions 1 and 2 (pyridazine), positions 1 and 3 (pyrimidine), or positions 1 and 4 (pyrazine). Preferably, the diazine used in the composition of the present invention is selected from the group of pyrimidines and pyrimidine derivatives comprising or consisting of thioguanine, fludarabine, cladribine, cytarabine, gemcitabine, 6-mercaptopurine, 5-fluorouracil (5-FU), and analogs thereof. Most preferably, the diazine used in the composition of the present invention is 5-FU or fludarabine.

[0083] According to one preferred embodiment, 5-FU is administered at a dose of 350 mg / m² to adult subjects. 2 ~400mg / m 2 It can be administered for 4-5 days every 4 weeks.

[0084] According to one preferred embodiment, fludarabine is administered orally at a dose of 40 mg / m². 2 It can be administered at a dose of 1 / day for 5 consecutive days every 28 days. The doses described herein are for humans, but can be adjusted to suit the size of other mammals and children, depending on weight or square meter size.

[0085] IUPAC defines transition metals as any element that has an incomplete subshell or can form stable ions only with an incomplete d subshell. Transition metals are the 40 chemical elements in the periods 21-30, 39-48, 71-80, and 103-112 of the periodic table. Preferably, the transition metal used in the composition of the present invention is selected from the group including or consisting of cisplatin, oxaliplatin, eptaplatin, lovaplatin, nedaplatin, carboplatin, iproplatin, satraplatin, tetraplatin, DCP, PLD-147, JM118, JM126, JM335, and other derivatives. Most preferably, the transition metal used in the composition of the present invention is carboplatin or cisplatin.

[0086] According to one preferred embodiment, cisplatin is administered to adult subjects at a dose of 50 mg / m². 2 ~100mg / m 2 It can be administered every 3 or 4 weeks. The dose for children is 60 mg / m² every 28 days. 2 That is the case.

[0087] According to one preferred embodiment, carboplatin is administered at a dose of 300 mg / m² to adult subjects. 2 ~400mg / m 2 It can be administered every 3 or 4 weeks. The dose for children is 200 mg / m² every 21 days. 2 (Kohler et al., European Journal of Cancer (2013) 49, 3671-3679).

[0088] The dosages described herein are for adult and / or child humans, but may be adjusted to suit the size of other mammals depending on their weight or square meter size.

[0089] Nitrogen mustard compounds are compounds that have two beta-haloalkyl groups bonded to a nitrogen atom. Among compounds in this class, cyclophosphamide is used to treat a variety of cancers.

[0090] According to one preferred embodiment, cyclophosphamide is administered at a dose of 400 mg / m² to adult subjects. 2 ~1800mg / m 2 It can be administered in divided doses over 2-5 days, and in the case of intermittent therapy, it can be repeated at intervals of 2-4 weeks, or in the case of daily continuous therapy, 60 mg / m². 2 ~120 mg / m² 2 It can be repeated daily. The dosage for children is 1.5 mg / m². 2 ~3mg / m 2 It is 5 days at a rate of / day.

[0091] The dosages described herein are for adult and / or child humans, but may be adjusted to suit the size of other mammals depending on their weight or square meter size.

[0092] According to one preferred embodiment, when using a composition comprising at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof to prevent and / or treat cancers expressing OAcGD2 ganglioside, selected from the group including or comprising neuroblastoma, glioma, retinoblastoma, Ewing family tumors, sarcomas (i.e., rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal cell carcinoma, head and neck cancer, and hematological cancers (i.e., leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and myeloma), at least one anticancer agent is selected from the group including or comprising cyclophosphamide, doxorubicin, topotecan, irinotecan, temozolomide (TMZ), retinoic acid (RA), 5-fluorouracil (5-FU), fludarabine, carboplatin, and cisplatin.

[0093] According to another preferred embodiment, when preventing and / or treating neuroblastoma using a composition comprising at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof, the anticancer agent is temozolomide, topotecan, irinotecan, fludarabine, cyclophosphamide, or a mixture thereof.

[0094] In this preferred embodiment, each of the claimed compounds (i.e., an antibody that recognizes OAcGD2 ganglioside and an anticancer agent) used in combination as a treatment for cancer expressing OAcGD2 ganglioside is separately known to have therapeutic activity against the same disease or related symptoms. Combining an antibody that recognizes OAcGD2 ganglioside with an anticancer agent produces unexpected technical effects, namely synergistic effects.

[0095] In one embodiment, at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is, a) Light chain variable region (VL) polypeptide of amino acid sequence SEQ ID NO: 1, and b) Heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 2 Includes.

[0096] The term "variable region" refers to the domains of the antibody heavy chain (VH) and light chain (VL) that are involved in the binding of antibodies to antigens.

[0097] According to one preferred embodiment, the light chain variable region (VL) polypeptide is the amino acid sequence SEQ ID NO: 3.

[0098] Preferably, the light chain variable region (VL) polypeptide is selected from the group comprising or consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0099] According to another preferred embodiment, the light chain variable region (VL) polypeptide has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs. 34, 35, 36, 37, 38, 39, and 40.

[0100] Preferably, the light chain variable region (VL) polypeptide has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs. 39 and SEQ ID NOs. 40.

[0101] According to another preferred embodiment, the heavy chain variable region (VH) polypeptide is amino acid sequence number 8.

[0102] Preferably, the heavy chain variable region (VH) polypeptide is selected from the group including or consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0103] According to another preferred embodiment, the heavy chain variable region (VH) polypeptide has an amino acid sequence selected from the group including or consisting of SEQ ID NOs: 41, SEQ ID NOs: 42, SEQ ID NOs: 43, SEQ ID NOs: 44, SEQ ID NOs: 45, SEQ ID NOs: 46, SEQ ID NOs: 47, SEQ ID NOs: 48, SEQ ID NOs: 49, SEQ ID NOs: 50, and SEQ ID NOs: 51.

[0104] Preferably, the heavy chain variable region (VH) polypeptide has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs. 48, SEQ ID NOs. 49, and SEQ ID NOs. 50.

[0105] According to another preferred embodiment, the light chain variable region (VL) polypeptide is amino acid sequence number 12.

[0106] According to another preferred embodiment, the heavy chain variable region (VH) polypeptide is amino acid sequence SEQ ID NO: 13.

[0107] More preferably, at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is a) The light chain variable region (VL) polypeptide of amino acid sequence SEQ ID NO: 14, and b) Heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 15 Includes.

[0108] Complementarity-determining regions (CDRs) of the heavy (VH) chain variable region having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and complementarity-determining regions of the light (VL) chain variable region having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0109] In another specific embodiment, at least one antibody, a functional fragment or derivative thereof that recognizes OAcGD2 ganglioside is, a) A heavy chain comprising three heavy chain complementary regions (CDRs) of amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and a heavy chain framework sequence derived from an immunoglobulin heavy chain, and b) A light chain comprising three light chain complementary regions (CDRs) of amino acid sequences SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and a light chain framework sequence derived from an immunoglobulin light chain. Includes.

[0110] The complementarity-determining regions (CDRs) occur at amino acid residues 26-35 (CDR1_VH), 50-68 (CDR2_VH), and 99-108 (CDR3_VH) of SEQ ID NO: 15, and at amino acid residues 24-39 (CDR1_VL), 54-60 (CDR2_VL), and 94-102 (CDR3_VL) of SEQ ID NO: 14.

[0111] The assignment of amino acids to each CDR follows well-known numbering systems, including the IMGT, Kabat, and Chothia systems (IMGT, The International Immunogenetics Information System®, LEFRANC et al., Nucleic Acids Research, vol.27, pp:209-212, 1999; KABAT, Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH publication, No.91-3242, 991; CLOTHIA & LESK, J Mol Biol., vol.196(4), pp:901-917, 1987).

[0112] The term "antibody that recognizes OAcGD2 ganglioside" is used when the temperature is 2 × 10⁶ at 25°C. 5Equilibrium binding constant (K) of antibodies against OAcGD2 gangliosides greater than M A ), preferably 2 × 10 6 K below M A , more preferably 1 × 10 7 M or more, or even 2 × 10 7 M or higher K A This refers to such affinity. This affinity can be easily measured by technologies available in the relevant field, such as scatchard analysis, competitive ELISA, BIACORE assay, or KINEXA assay.

[0113] According to the present invention, the antibody used in the composition according to the present invention is not specific to GD2 ganglioside. The equilibrium binding constant (K) of the antibody used in the composition according to the present invention A The ratio of ) to GD2 ganglioside is at least 1 / 10, more preferably at least 1 / 100, compared to OAcGD2 ganglioside. Preferably, 10 at 25°C 5 Equilibrium binding constant (K) of antibodies against GD2 gangliosides less than M A Preferably 10 units relative to GD2 ganglioside 4 Less than M K A .

[0114] The term "functional fragment" refers to antibody fragments that specifically bind to the OAcGD2 ganglioside. Such fragments can be easily identified by those skilled in the art and include, as examples, scFv fragments, Fab fragments (e.g., by papain digestion), Fab' fragments (e.g., by pepsin digestion and partial reduction), F(ab')2 fragments (e.g., by pepsin digestion), Fabc (e.g., by plasmin digestion), and Fv and Fd (e.g., by pepsin digestion, partial reduction and re-aggregation) fragments, which are encompassed by the present invention.

[0115] These fragments can be produced by enzymatic cleavage, synthesis, or recombination techniques using methods well known in the art, such as those described by STANWORTH et al. (Handbook of Experimental Immunology, vol. 1, chapter 8, Blackwell Scientific Publications, 1978). Antibodies can also be produced using antibody genes in various cleavage forms, with one or more stop codons introduced upstream of the native termination site. For example, a combination gene encoding the F(ab')2 double-chain portion can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. Various parts of the antibody can be chemically linked by conventional techniques or prepared as adjacent proteins using genetic engineering techniques.

[0116] Here, these fragments include at least the variable regions of the heavy and light chains mentioned above.

[0117] These fragments can be made soluble, but they can also be immobilized within the cell membrane as single-chain variable regions of chimeric antigen receptors (CARs).

[0118] In this specification, the term “chimeric antigen receptor (CAR)” refers to an artificial hybrid polypeptide comprising at least one antigen-binding domain and at least one effector cell signaling domain of an antibody. Such CARs encompass engineered receptors that imprint artificial specificity onto specific immune effector cells (e.g., T cells, NK cells, and NKT cells). CARs can be used to confer the specificity of monoclonal antibodies onto T cells in a manner that is not constrained by MHC, thus leveraging the antigen-binding properties of monoclonal antibodies. When expressed in T cells, CARs recognize untreated antigens independently of their expression of major histocompatibility antigens, distinct from physiological T cell receptors (TCRs), and thus bypass two major mechanisms of tumor escape: downregulation of HLA expression or proteasomal antigen processing. Binding of CARs to specific antigens triggers an immune response.

[0119] In certain embodiments, the CAR comprises an outer domain, a transmembrane domain, and an inner domain, where the sequence may be a bispecific antibody or a multi-linked chimeric receptor (e.g., the multi-linked chimeric antigen receptor described in International Publication No. 2016 / 016343).

[0120] The external domains correspond to the antigen-binding domain and the spacer domain (stalk region). The antigen-binding domain is preferably a single-chain variable fragment (scFv). Such scFvs are genetically engineered antibody fragments, usually consisting of the heavy and light chains of immunoglobulins, or parts thereof such as VH, VL, linked by a flexible peptide linker, as disclosed as an example in PLUCKTHUN (The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315, 1994). The flexible peptide linker may be a peptide of 6-40 amino acid residues. The use of small amino acids such as alanine and glycine is useful for creating flexible linkers. An example of a flexible linker is glycine polymer (G)n, e.g., (GS) n GSGGS n (Sequence code 22)n, GGGS n (Sequence No. 23)n and GGGGS nExamples of useful polymers include glycine-serine polymers such as (SEQ ID NO: 24)n, where n is at least an integer of 1, glycine-alanine polymers or glycine-serine polymers, or other flexible linkers known in the art. Examples of useful polymers include GGGGSGGGGSGGGGS ((G4S)3; SEQ ID NO: 25), GTSTGSGKPGSGEGSTKG (CD19 linker; SEQ ID NO: 26), GGSSRSSSSGGGGSGGGG (18-mer; SEQ ID NO: 27), GGGGSGGGGSGGGGSGGGGS ((G4S)4; SEQ ID NO: 28), KESGSVSSEQLAQFRSLD (SEQ ID NO: 29), EGKSSGSGSESKST (SEQ ID NO: 30), GSAGSAAGSGEF (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), or GGGGGG (SEQ ID NO: 33). Finally, these scFv fragments can be obtained by methods well known to those skilled in the art, such as those described by GILLILAND et al. (Tissue Antigens, vol. 47, pp. 1-20, 1996). In this specification, the term “stalk region” is also called a “spacer or hinge domain” and refers to any oligo or polypeptide that functions to link a transmembrane domain to an external domain. In particular, stalk regions are used to confer greater flexibility and reachability to the external domain. Spacer sequences play a primarily structural role in CARs. Spacers physically separate the target region from the T cell membrane. The optimal distance required may differ for each antigen. Spacers are thought to be necessary when CARs bind to epitopes close to the target cell membrane to enable efficient target approach, or when the size and glycosylation state of the antigen are complex. Human IgG-derived spacers (hinge-CH2-CH3) are commonly used for their stabilizing effect on CAR expression; however, the interaction between the spacer's Fc domain and the Fc gamma receptor (FcgR) on bone marrow cells can lead to activation-induced cell death of T cells, potentially limiting their survival in vivo. This can be overcome by removing or modifying the constant heavy chain (CH)2 domain region, which is essential for FcgR binding, thereby improving CAR T cell survival and antitumor activity in preclinical models.Other commonly used hinge domains include those derived from CD28 or CD8, or other cleavage fragments from human IgG-derived spacers. In one preferred embodiment, the CAR includes a stalk region between the external domain and the transmembrane domain. The stalk region may contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. This stalk region may be derived from all or part of a native molecule, such as a portion of the extracellular region of CD8, CD4, or CD28, or from all or part of the antibody constant region. Alternatively, this stalk region may be a synthetic sequence.

[0121] The transmembrane domain is a membrane anchor domain and also a linker between the outer and inner domains. This transmembrane domain may be the human IgG4Fc hinge region, Fc region, CD4 transmembrane domain, T cell receptor transmembrane domain, or other transmembrane domains derived from other human transmembrane signaling proteins such as CD16, TCR zeta chain (CD3ζ), CD28 and CD8 and erythropoietin receptor, or their variants. Preferably, this transmembrane domain is the T cell receptor transmembrane domain. Preferably, the T cell receptor transmembrane domain is derived from a transmembrane protein that can form a complex with the T cell receptor (TCR) of an antigen. Preferably, the T cell receptor transmembrane domain includes one or more of the following, in whole or in part: TCR zeta chain (CD3ζ), CD28, OX40 / CD134, 4-1BB / CD137 / TNFRSF9, FcεRIγ, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, CD27, DAP10, and CD40.

[0122] The internal domain is an intracellular signaling domain, which is responsible for intracellular signaling after the binding of the external domain to a target antigen that activates immune cells. In other words, the intracellular signaling domain is responsible for the activation of at least one normal effector function of the immune cell on which the chimeric receptor is expressed. The term "effector function" refers to the specific function of a T cell, which may be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and induces the cell to perform its specific function. Usually, the entire intracellular signaling domain is used, but in many cases, it is not necessary to use the entire intracellular polypeptide. To the extent that cleavage fragments of the intracellular signaling domain can be used, these cleavage fragments can be used in place of the complete chain, as long as they still transmit effector function signals. The term intracellular signaling domain therefore means that it contains any cleavage fragment of the intracellular signaling domain sufficient to transmit effector function signals.

[0123] Preferred examples of signaling domains used in multi-chain CARs may be cytoplasmic sequences of Fc receptors or T cell receptors and co-receptors that work in conjunction to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities. The signaling domains include two distinct classes of cytoplasmic signaling sequences: those that induce antigen-dependent primary activation and those that act antigen-independently to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence may include signaling motifs known as the immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well-defined signaling motifs found at the intracytoplasmic terminals of various receptors that act as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention, non-limiting examples, include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one preferred embodiment, the signaling domain of the multi-chain CAR may include a CD3 zeta signaling domain or an intraplasmic domain of an Fc epsilon RI beta or gamma chain.

[0124] In certain embodiments, the signaling domain of the multi-chain CAR of the present invention includes a co-stimulatory signaling molecule. The co-stimulatory molecule is a cell surface molecule other than an antigen receptor or a ligand thereof that is necessary for an efficient immune response.

[0125] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a co-stimulatory molecule on a T cell, thereby providing signals that mediate the T cell response, including, but not limited to, the primary signal provided by the binding of the TCR / CD3 complex with MHC molecules containing peptides, as well as proliferation activation and differentiation. Examples of costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), cell adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands include, but are not limited to, ligands that specifically bind to CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, DAP-10, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83, as well as antibodies that specifically bind to costimulatory molecules present on T cells.

[0126] A "costimulatory molecule" refers to a congeneral binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the cell, including but not limited to proliferation. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, DAP-10, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0127] The term "derivative" refers to an amino acid sequence having a percentage of identity of at least 90%, preferably at least 95%, most preferably at least 98% (i.e., corresponding to about 12, 6, and 2 amino acid substitutions, respectively), and preferably at least 99% (i.e., corresponding to about 1 amino acid substitution) with an amino acid sequence selected from the group including or consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51.

[0128] In relation to CDR sequences, the term derivative refers to an amino acid sequence having at least 80%, preferably at least 90% (i.e., corresponding to about two and one amino acid substitutions, respectively) of identity with an amino acid sequence selected from the group including or consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0129] Such derivatives can be readily identified by those skilled in the art, taking into account their personal knowledge and the teachings of this patent application. It is also understood that natural amino acids can be replaced with chemically modified amino acids. Generally, such chemically modified amino acids increase the polypeptide half-life.

[0130] In this specification, the “percentage of identity” between two amino acid sequences refers to the percentage of identical amino acids obtained by the best alignment of the sequences between the two sequences being compared. This percentage is purely statistical, and the differences between these two sequences are randomly distributed across the amino acid sequences. In this specification, “best alignment” or “optimal alignment” refers to the alignment that yields the highest desired percentage of identity (see below). Sequence comparisons between two amino acid sequences are typically performed by comparing these sequences that have been pre-aligned according to the best alignment. This comparison is performed on a comparison segment to identify and compare local regions of similarity. The best sequence sorting for comparison can be done manually, using local homology algorithms developed by Smith and Waterman (Ad.App.Math., vol.2, p:482, 1981), global homology algorithms developed by Neddleman and Wunsch (J.Mol.Biol., vol.48, p:443, 1970), similarity methods developed by Pearson and Lipmolan (Proc.Natl.Acad.Sci.USA, vol.85, p:2444, 1988), computer software using these algorithms (Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA, GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA), and MUSCLE multiple sorting algorithms (Edgar, Robert C., Nucleic Acids). This can be carried out using Research, vol.32, p:1792, 2004). To obtain the best local alignment, BLAST software with a BLOSUM62 matrix can preferably be used. The degree of identity between two amino acid sequences is determined by comparing these two optimally aligned sequences, and the amino acid sequences may include additions or deletions to the reference sequence in order to obtain the best alignment between these two sequences.The percentage of identity is calculated by determining the number of identical positions between the two sequences, dividing this number by the total number of positions to be compared, and multiplying the result by 100 to obtain the percentage of identity between the two sequences.

[0131] The antibodies used in the compositions of the present invention are produced by recombinant DNA.

[0132] The antibody used in the composition of the present invention may or may not be glycosylated, but glycosylated antibodies are preferred. In one preferred embodiment, the antibody used in the composition of the present invention can be low in fucose.

[0133] The antibody used in the composition of the present invention can be an immune complex.

[0134] In this specification, the term “immune complex” refers to a complex molecule comprising a second molecule, preferably an immunomodulator, a cytotoxic drug, or at least one antibody conjugated to a radioisotope, a functional fragment thereof, or a derivative thereof. Such an immune complex may be an antibody-drug conjugate (ADC), an immune cytokine (ICK), or an antibody-radioconjugate (ARC). Here, the second molecule may be an antibody having binding specificity to another antigen, and the formed immune complex may be a bispecific antibody such as a BiTE (bispecific T cell conjugate). The antibody or fragment thereof complexes or covalently binds to the second molecule (e.g., a fusion protein). Preferably, the antibody or fragment thereof binds to the second molecule by covalent bond. The second molecule may be a protein or glycoprotein that can specifically interact with sugars to form non-covalent bonds such as lectins.

[0135] The antibodies used in the compositions of the present invention may be bispecific or multispecific antibodies. Several forms, such as bivariable domain immunoglobulins (DVDs), bispecific T cell conjugates (BiTEs), bispecific antibodies, tetravalent tandem antibodies (TandAbs), or biaffinity retargeting molecules (DARTs), are suitable forms of antibodies used in the compositions (WEIDLE et al., Cancer Genomics & Proteomics, 2013, vol.10, pp.1~18).

[0136] Preferably, when the patient is human, the antibody used to treat cancer expressing OAcGD2 ganglioside is a human or humanized (CDR graft) version of the antibody, although a mouse version of the antibody may also be used. When considering repeated treatment, human or humanized IgG antibodies are less likely to generate an anti-IgG immune response from the patient.

[0137] In all embodiments, the composition according to the present invention comprises a therapeutically effective amount of at least one anticancer agent and a therapeutically effective amount of at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof.

[0138] According to one preferred embodiment, an antibody, its functional fragment, or derivative is injected into a target of 1 m 2 Dosage per unit: 2-2,000 mg, preferably 5-1,000 mg / m² 2 Most preferably, the dose is 10-500 mg / m². 2 It can be administered as follows.

[0139] The dosages described herein are for humans, but can be adjusted to suit the size of other mammals and children, depending on weight or square meter size.

[0140] In this specification, the term “therapeutic dose” encompasses any amount that yields the desired therapeutic or biological effect. The therapeutic effect depends on the cancer expressing the OAcGD2 ganglioside being treated or the desired biological effect. Therefore, the therapeutic effect may be a reduction in the severity of symptoms associated with cancer expressing the OAcGD2 ganglioside, and / or (partial or complete) inhibition of the progression of cancer expressing the OAcGD2 ganglioside. The amount required to induce a therapeutic response can be determined based on the type of cancer, the patient’s age, health, physique, and sex.

[0141] The optimal amount can also be determined based on monitoring the patient's response to the treatment.

[0142] In one embodiment, the composition also includes a pharmaceutically acceptable carrier used in therapy.

[0143] The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically acceptable and do not generally cause allergic reactions or similar undesirable reactions such as stomach upset or dizziness when administered to humans. Preferably, as used herein, "pharmaceutically acceptable" means that it is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for animals, or more specifically for humans.

[0144] The term "carrier" refers to a solvent, adjuvant, excipient, or vehicle to which a compound is administered. Such drug carriers can be sterile liquids such as water and oil, and may include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil.

[0145] The compositions may be in any dosage form suitable for administration to a patient, including but not limited to solutions, suspensions, lyophilized powders, capsules, and tablets. Here, the route of administration of the compositions of the present invention is preferably parenteral, and as used herein, the term “parenteral” includes intravenous, intramuscular, subcutaneous, intraperitoneal, rectal, vaginal, mucosal, intrathecal, intracranial, or intratumoral administration. Accordingly, the pharmaceutical compositions include a pharmaceutically acceptable vehicle for the formulation to be injected. These may be, in particular, isotonic sterile saline (such as monosodium phosphate or disodium phosphate, sodium chloride, potassium, calcium, or magnesium, or mixtures of such salts), or dry, especially lyophilized, compositions to which sterile water or physiological saline may be added as needed to constitute the injection solution. Suitable drug carriers are described in “Remington's Pharmaceutical Sciences” by EW Martin.

[0146] Most preferably, the composition is in any dosage form suitable for intravenous administration to a patient.

[0147] The antibodies, functional fragments, or derivatives of the present invention can be solubilized in a buffer or water, or encapsulated in emulsions, microemulsions, hydrogels (e.g., hydrogels based on PLGA-PEG-PLGA triblock copolymer), microspheres, nanospheres, fine particles, nanoparticles (e.g., poly(lactic acid-coglycolic acid) fine particles (e.g., polylactic acid (PLA); poly(lactide-coglycolic acid) (PLGA); polyglutamic acid microspheres, nanospheres, fine particles, or nanoparticles)), liposomes, or other Galenic formulations. In all cases, the formulations must be sterile and fluid enough to be used in a syringe. The formulations must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi.

[0148] The dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0149] The antibodies, functional fragments, or derivatives of the present invention can be formulated in compositions in the form of neutral or salt. Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid and phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid (formed with the free amino group of the protein). Salts formed with the free carboxyl group can also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium, or iron(III), and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0150] The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The antibodies of the present invention may also be modified, for example, by PEGylation, to enhance their bioavailability.

[0151] Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Microbial activity can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride.

[0152] The absorption of an injectable composition can be extended by using absorption-delaying agents, such as aluminum monostearate, gelatin, polyols, and half-life-extending covalent and non-covalent formulations, in the composition.

[0153] Numerous causes of peptide instability or degradation exist, including hydrolysis and denaturation. Hydrophobic interactions can lead to molecular aggregation (i.e., clumping). These problems can be reduced or prevented by adding stabilizers.

[0154] Examples of stabilizers include cyclodextrins and their derivatives (see U.S. Patent No. 5,730,969). The final formulation can also be stabilized by adding appropriate preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran, and glycerin. Stabilizers selected from ionic and nonionic surfactants, D-glucose, D-galactose, D-xylose, D-galacturonic acid, trehalose, dextran, hydroxyethyl starch, and mixtures thereof can be added to the formulation. The addition of alkali metal salts or magnesium chloride can stabilize peptides. Peptides can also be stabilized by contacting them with sugars selected from the group consisting of dextran, chondroitin sulfate, starch, glycogen, dextrin, and alginates. Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (e.g., glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). Polyols can stabilize peptides and are either water-miscible or water-soluble. Suitable polyols can be polyhydroxy alcohols, monosaccharides, and disaccharides, including mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, vinylpyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and their polymers. Various excipients, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinylpyrrolidone, hydrolyzed gelatin, and ammonium sulfate, can also stabilize peptides.

[0155] In all embodiments, at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, and at least one antibody that recognizes OAcGD2 ganglioside, its functional fragment, or derivative can be administered separately, simultaneously, or sequentially.

[0156] In one embodiment, at least one anticancer agent, administered separately, simultaneously, or sequentially with at least one antibody, functional fragment, or derivative that recognizes OAcGD2 ganglioside, has a molecular weight of 10 kDa to 15 kDa.

[0157] Furthermore, in one embodiment, at least one anticancer agent administered separately, simultaneously, or sequentially with at least one antibody, functional fragment, or derivative that recognizes OAcGD2 ganglioside has a molecular weight of 80 kDa to 200 kDa.

[0158] Furthermore, in one embodiment, at least one anticancer agent administered separately, simultaneously, or sequentially with at least one antibody, functional fragment, or derivative that recognizes OAcGD2 ganglioside has a molecular weight of 120 to 800 daltons.

[0159] For example, at least one antibody, functional fragment, or derivative thereof that recognizes OAcGD2 ganglioside can be administered before the administration of at least one anticancer agent with a molecular weight of 100 to 200,000 daltons. The method and sequence of administration can vary in duration, from rapid total dose to continuous infusion, with the aim of maximizing the efficacy of the combination. As a result, the combination of at least one anticancer agent with a molecular weight of 100 to 200,000 daltons and at least one antibody, functional fragment, or derivative thereof that recognizes OAcGD2 ganglioside is not limited to those obtained by their physical binding, nor does it allow for separate administration, which may be simultaneous or sequential.

[0160] In some embodiments, the pharmaceutical composition includes, but is not limited to, alkylating agents, antimetabolites, antitumor antibodies, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors, corticosteroids, hormones or hormone-like drugs, cytokines, nucleoside analogs, and second, third, fourth, and nth anticancer agents.

[0161] An example of another anticancer agent that can be used in a method for treating and / or preventing cancer expressing OAcGD2 ganglioside, administered separately or in the same composition, is a composition comprising: (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons; (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof; and optionally (iii) a pharmaceutically acceptable carrier. (a) Alkylating agents: mechloramine, chlorambucil, ifosfamide, melphalan, etc. (b) Antimetabolites: 6-mercaptopurine (6-MP), capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, etc. (c) Antitumor antibiotics: Actinomycin-D, bleomycin, mitomycin-C, mitoxantrone, etc. (d) Topoisomerase inhibitors: etoposide (VP-16), teniposide, etc. (e) Mitotic inhibitors: Paclitaxel, docetaxel, etc. (f) Tyrosine kinase inhibitors: Imatinib, gefininib, sunitinib, etc. (g) Corticosteroids: Prednisone, methylprednisone, dexamethasone, etc. (h) Hormones or hormone-like drugs: fulvestrant, tamoxifen, toremifene, anastrozole, exemestane, letrozole, megestrol acetate, estrogen, bicalutamide, flutamide, nilutamide, leuprolide, goserelin, etc. (i) Cytokines: Soluble small proteins of about 5-20 kDa that are released by a single cell population (e.g., first-stimulated T lymphocytes) upon contact with a specific antigen and act as intercellular mediators. Examples of cytokines include lymphokines, monokines, interleukins, and several related signaling molecules such as tumor necrosis factor (TNF) and interferons. (j) Nucleoside analogs: cladribine, fludarabine, pentostatin, etc. These include, but are not limited to, those listed above.

[0162] In some embodiments, the pharmaceutical composition includes, but is not limited to, nucleic acids, such double-stranded synthetic short RNA molecules (miRNAs), or synthetic DNA / RNA-like oligonucleotides (ASOs), as well as second, third, fourth, and nth anticancer agents. MicroRNAs (miRNAs) are 16-25 nucleotide endogenous single-stranded non-coding RNAs involved in post-transcriptional decay of mRNA translation. Their deregulation is frequently observed in numerous diseases, including cancer. The importance of the mir-34 family and mir-17-92 cluster has been established in both tumorigenesis and metastasis in neuroblastoma, while overexpression of mir-184 reduces neuroblastoma tumor growth (Chu and Lee, 2012, Intech, MicroRNA target signatures in Advanced stage Neuroblastoma, Neuroblastoma-Present and future, Prof. Hiroyuki Shimada, IBSN 978-953-307-016-2, Chapter 13, pp. 271-286). Therefore, by designing various types of small single- or double-stranded chemosynthetic and optimized nucleic acids (from about 22 nucleotides), such as miRNA mimes or agomyl that upregulate miRNA activity, or miRNA inhibitors or antagomils that knock down individual miRNA molecules, it is possible to control specific miRNA activity or to strictly regulate miRNA cellular levels in cells. Such short chemosynthetic nucleic acids targeting cancer-related miRNAs expressing OAcGD2 gangliosides can be used in the compositions according to the present invention.

[0163] A second aspect of the present invention relates to a method for enhancing the efficacy of treating cancer expressing OAcGD2 ganglioside, comprising the steps of administering to a patient in need an effective amount of a composition comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0164] In one embodiment, in a composition used to enhance the effectiveness of treating cancer expressing OAcGD2 ganglioside, at least one anticancer agent has a molecular weight of 10 kDa to 15 kDa.

[0165] Furthermore, in one embodiment, in a composition used in a method to enhance the efficacy of treating cancer expressing OAcGD2 ganglioside, at least one anticancer agent has a molecular weight of 80 kDa to 200 kDa.

[0166] Furthermore, in one embodiment, in a composition used in a method for enhancing the efficacy of treating cancer expressing OAcGD2 ganglioside, at least one anticancer agent has a molecular weight of 120 to 800 daltons.

[0167] The phrase "enhancing the effectiveness of the treatment" refers to the increase in the number of cancer cell deaths obtained by the method of the present invention compared to treatment using only one anticancer drug.

[0168] The efficacy of anticancer drugs with molecular weights of 100 to 200,000 daltons can be significantly improved when administered in combination with at least one antibody that recognizes OAcGD2 ganglioside, which has a different mechanism of action than anticancer drugs with molecular weights of 100 to 200,000 daltons, or its functional fragment or derivative.

[0169] The improved efficacy of the combination according to the present invention can be demonstrated by seeking therapeutic synergies. A clear and well-established definition of "synergy" is that the effect of the combined drugs is greater than the simple additive effect that would be individually expected from knowledge of the effects of each drug. Possible favorable consequences of synergy can enhance the efficacy of the therapeutic effect. Preferably, cancers expressing OAcGD2 ganglioside are more effectively treated when the composition of the present invention is used.

[0170] The efficacy of combinations of at least one anticancer drug with a molecular weight of 100 to 200,000 daltons and at least one antibody that recognizes OAcGD2 ganglioside, or its functional fragment or derivative, can be interpreted from experimental data using the Combination Index (CI) method developed by Chou and Talalay (Chou TC, Pharmacol Rev 58:621-681, 2006). Briefly speaking, in the case of a combination of two drugs (D)1 and (D)2 with a combination ratio of (D)1:(D)2=P:Q, the combination (D) 1,2 In =(D)1+(D)2, the inventors have found that (D)1=(D) 1,2 ×[P / (P+Q)] and (D)²=(D) 1,2 We obtain ×[Q / (P+Q)]. Combinatorial exponential formula CI x =[(D)1 / (D x )1]+[(D)2 / (D x According to the results of [2], x% inhibition of the system D x (F a ) x Given the effect, the combined additive effect on the sum of the divided doses of each drug is (D)1 / (D x )1 and (D)2 / (D x )² should be equal to 1. CI=1 indicates additive effect, CI<1 indicates synergistic effect, and CI>1 indicates antagonistic effect.

[0171] The effectiveness of a combination against solid tumors can also be experimentally determined, for example, by comparing numerical values ​​of tumor size before and after treatment. Preferably, a combination of at least one anticancer agent with a molecular weight of 100 to 200,000 daltons disclosed herein and at least one antibody that recognizes OAcGD2 ganglioside, its functional fragment, or derivative is effective if it reduces tumor size by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%, and more preferably at least 50-70%.

[0172] The effectiveness of a combination of therapies for solid tumors can also be determined, for example, by tracking the number of cancer cells, cancer cell infiltration into peripheral organs, or tumor metastasis.

[0173] The effectiveness of cancer treatment can also be measured by evaluating the overall survival, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life of patients with cancer expressing OAcGD2 ganglioside.

[0174] In a preferred embodiment, at least one anticancer agent of the composition according to the present invention, having a molecular weight of 100 daltons to 200,000 daltons, is the same as the one initially selected to treat cancer expressing OAcGD2 ganglioside as a single agent.

[0175] Another possible and advantageous outcome of the synergistic effect could be a reduction in the dosage of at least one anticancer drug, while simultaneously increasing or maintaining the same efficacy to avoid toxicity.

[0176] A third aspect of the present invention relates to a method for enhancing sensitivity to an anticancer agent in a patient with cancer expressing OAcGD2 ganglioside, the method comprising the steps of administering to a patient in need an effective amount of a composition comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0177] In one embodiment, a composition used in a method for enhancing sensitivity to anticancer drugs in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer drug has a molecular weight of 10 kDa to 15 kDa.

[0178] Furthermore, in one embodiment, a composition used in a method for enhancing sensitivity to anticancer drugs in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer drug has a molecular weight of 80 kDa to 200 kDa.

[0179] Furthermore, in one embodiment, a composition used in a method for enhancing sensitivity to anticancer drugs in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer drug has a molecular weight of 120 to 800 daltons.

[0180] In addition to providing improved treatment for cancer, the administration of the combinations described herein can improve the quality of life of patients compared to the quality of life experienced by the same patients receiving different treatments. For example, the administration of a combination of at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside as described herein and an anticancer drug to patients in need can provide an improved quality of life compared to the quality of life that the same patient would experience if they received anticancer drugs alone as therapy. For example, combination therapy with the combinations described herein can reduce the required dose of anticancer drugs, thereby reducing treatment-related side effects (e.g., nausea, vomiting, hair loss, rash, loss of appetite, etc.). The combinations can also reduce tumor burden and associated adverse events such as pain, organ damage, and weight loss.

[0181] Another possible benefit of the synergistic effect is that it may minimize or delay the development of drug resistance and its dramatic consequences, such as the recurrence of cancers expressing the OAcGD2 ganglioside.

[0182] A fourth aspect of the present invention relates to a method for preventing or delaying the development of anticancer drug-resistant cancer in a patient with cancer expressing OAcGD2 ganglioside, the method comprising the steps of administering to a patient in need an effective amount of a composition comprising (i) at least one anticancer drug having a molecular weight of 100 to 200,000 daltons, (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and optionally (iii) a pharmaceutically acceptable carrier.

[0183] In one embodiment, a composition used in a method for preventing or delaying the development of anticancer drug-resistant cancer in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer agent has a molecular weight of 10 kDa to 15 kDa.

[0184] Furthermore, in one embodiment, a composition used in a method for preventing or delaying the development of anticancer drug-resistant cancer in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer agent has a molecular weight of 80 kDa to 200 kDa.

[0185] Furthermore, in one embodiment, a composition used in a method for preventing or delaying the development of anticancer drug-resistant cancer in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer agent has a molecular weight of 120 to 800 daltons.

[0186] It is not unusual for patients to survive for a short period after primary cancer treatment with a single anticancer drug (i.e., first-line cancer treatment) without any signs or symptoms of cancer before the recurrence of cancer or signs and symptoms of cancer. The objective is to prevent clonal selection that leads to the proliferation of resistant cells by using a combination of (ii) at least one anticancer drug with a molecular weight of 100 to 200,000 daltons and (ii) at least one antibody that recognizes OAcGD2 ganglioside, its functional fragment or derivative.

[0187] In one preferred embodiment, at least one anticancer agent of the composition according to the present invention, having a molecular weight of 100 daltons to 200,000 daltons, is different from the initial stage of cancer resistance.

[0188] The mechanisms of treatment resistance include increased recognition and repair of DNA damaged by anticancer drugs, altered cell cycle checkpoint control, dysfunction of apoptotic pathways, and reduced drug accumulation as a result of increased expression of ABC transporters that leach anticancer drugs. Evidence is emerging that cancer stem cells (CSCs) are a subpopulation of cells within cancer characterized by increased resistance to chemotherapy and radiotherapy, indicating that conventional anticancer methods often fail to eradicate the cell subset that induces and perpetuates tumorigenesis. Therefore, cancer cells other than CSCs may increase their resistance to chemotherapy and radiotherapy, possibly by utilizing similar pathways. Reversal of chemotherapy resistance can be achieved by specific blockade of multidrug-resistant ABC transporters, as demonstrated in human melanoma.

[0189] Remarkably, the inventors have identified a novel method to reverse chemotherapy resistance, particularly in the CSC population, without requiring interference with multidrug resistance transporters or shuttles involved in anticancer drug uptake.

[0190] This fourth aspect of the present invention relates to a method for treating refractory or recurrent cancer in a patient with cancer expressing OAcGD2 ganglioside, comprising the step of administering to a patient in need an effective amount of a composition comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons and (ii) at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside, wherein the cancer is refractory to or subsequently recurs in response to first-line cancer treatment comprising one or more anticancer agents selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors and nucleoside analogs.

[0191] This fourth aspect of the present invention also relates to a composition for use in treating refractory or recurrent cancer in patients with cancer expressing OAcGD2 ganglioside, comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons and (ii) at least one antibody, functional fragment or derivative thereof that recognizes OAcGD2 ganglioside, wherein the cancer is refractory to or subsequently recurs in first-line cancer treatment comprising one or more anticancer agents selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors and nucleoside analogs.

[0192] In one embodiment, a composition used for a method of treating refractory or recurrent cancer in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer agent has a molecular weight of 10 kDa to 15 kDa.

[0193] Furthermore, in one embodiment, a composition used for a method of treating refractory or recurrent cancer in patients with cancer expressing OAcGD2 ganglioside, wherein at least one anticancer agent has a molecular weight of 80 kDa to 200 kDa.

[0194] Furthermore, in one embodiment, in a composition used for a method of treating refractory or recurrent cancer in patients with cancer expressing OAcGD2 ganglioside, at least one anticancer agent has a molecular weight of 120 to 800 daltons.

[0195] Furthermore, in one embodiment, the refractory or recurrent cancer is neuroblastoma.

[0196] According to another preferred embodiment, when the composition is used in a method for treating refractory or recurrent neuroblastoma, at least one anticancer agent is temozolomide, topotecan, irinotecan, cyclophosphamide, fudarabine, cisplatin, doxorubicin, isotretinoin, etoposide, or a mixture thereof.

[0197] Furthermore, in one embodiment, the refractory or recurrent cancer is glioblastoma.

[0198] According to another preferred embodiment, when the composition is used in a method for treating refractory or recurrent glioblastoma, at least one anticancer agent is temozolomide, topotecan, irinotecan, cyclophosphamide, fludarabine, cisplatin, carboplatin, or a mixture thereof.

[0199] In a preferred embodiment, at least one anticancer agent with a molecular weight of 100 to 200,000 daltons in the composition used for treating refractory or recurrent cancer is the same as that used for first-line cancer treatment.

[0200] Another possible and advantageous outcome of synergy may be the provision of synergistic combinations that rely on a balance between synergy and cytotoxicity.

[0201] A fifth aspect of the present invention is an in vitro / ex vivo method for identifying synergistic combinations of (ii) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons and (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof, a) Incubating primary tumor cells or cancer cell lineage cells expressing an O-acetylated form of GD2 ganglioside in vitro with a composition comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons and (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment or derivative thereof, and b) A step in which the CI50 index is measured according to the method disclosed by Chou and Talalay, and a CI50 of less than 1 represents synergy. The method includes the above.

[0202] Primary tumor cells are, for example, explanted human tumor cells. Any derivative of these cells can give rise to, for example, cancer cell lines.

[0203] In one embodiment, in the synergistic combination identified by the in vitro / ex vivo method of the present invention, at least one anticancer agent has a molecular weight of 10 kDa to 15 kDa.

[0204] Furthermore, in one embodiment, in the synergistic combination identified by the in vitro / ex vivo method of the present invention, at least one anticancer agent has a molecular weight of 80 kDa to 200 kDa.

[0205] Furthermore, in one embodiment, in the synergistic combination identified by the in vitro / ex vivo method of the present invention, at least one anticancer agent has a molecular weight of 120 to 800 daltons.

[0206] A sixth aspect of the present invention relates to an in vitro screening kit comprising (ii) a synergistic combination of at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons and (ii) at least one antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof.

[0207] In one embodiment, the synergistic combination in vitro screening kit of the present invention has at least one anticancer agent having a molecular weight of 10 kDa to 15 kDa.

[0208] Furthermore, in one embodiment, the synergistic combination in vitro screening kit of the present invention includes at least one anticancer agent having a molecular weight of 80 kDa to 200 kDa.

[0209] Furthermore, in one embodiment, the synergistic combination in vitro screening kit of the present invention includes at least one anticancer agent having a molecular weight of 120 to 800 daltons.

[0210] A seventh aspect of the present invention relates to a kit suitable for treating cancer expressing OAcGD2 ganglioside. In all embodiments, the kit comprises (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, (ii) at least one antibody, a functional fragment or derivative thereof that recognizes OAcGD2 ganglioside, and optionally (iii) a pharmaceutically acceptable carrier.

[0211] In one embodiment, in a kit suitable for treating cancer expressing the OAcGD2 ganglioside of the present invention, at least one anticancer agent has a molecular weight of 10 kDa to 15 kDa.

[0212] Furthermore, in one embodiment, in a kit suitable for treating cancer expressing the OAcGD2 ganglioside of the present invention, at least one anticancer agent has a molecular weight of 80 kDa to 200 kDa.

[0213] Furthermore, in one embodiment, in a kit suitable for treating cancer expressing the OAcGD2 ganglioside of the present invention, at least one anticancer agent has a molecular weight of 120 to 800 daltons.

[0214] The device may also include a mechanism capable of delivering kit components via a specific, selected route of administration.

[0215] The kit components can be packaged together or divided into two or more containers. In some embodiments, the containers may be vials containing sterile lyophilized formulations of compositions suitable for reconstitution. The kit may also include one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that can be used include, but are not limited to, pouches, trays, boxes, and tubes. The kit components can be packaged and maintained sterile within the container. Another component that may be included is instructions for the kit user on how to use it.

[0216] Antibodies that recognize OAcGD2 ganglioside, their functional fragments, or derivatives are useful as adjuvants in antitumor therapy.

[0217] An eighth aspect of the present invention relates to the use of an antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof, for enhancing the intracellular uptake of an anticancer agent with a molecular weight of 100 to 200,000 daltons in cells expressing an O-acetylated form of GD2 ganglioside.

[0218] In one embodiment, an antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is used to enhance the intracellular uptake of an anticancer agent with a molecular weight of 10 kDa to 15 kDa.

[0219] Furthermore, in one embodiment, an antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is used to enhance the intracellular uptake of anticancer drugs with molecular weights of 80 kDa to 200 kDa.

[0220] Furthermore, in one embodiment, an antibody that recognizes OAcGD2 ganglioside, a functional fragment thereof, or a derivative thereof is used to enhance the intracellular uptake of anticancer drugs with a molecular weight of 120 to 800 daltons.

[0221] The following embodiments are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention, but should not be construed as limiting their scope. [Examples]

[0222] 1. Treatment of neuroblastoma and glioblastoma cells with anti-OAcGD2mAb8B6 induces pore formation within the cell membrane. Culture dishes (6 wells) were coated with 500,000 IMR5, LAN-1, or DUASOII cells / well 24 hours prior to incubation with anti-OAcGD2mAb8B6 (mouse, IgG3) or isotype-matched negative control mouse IgG3 (CTRL-). Then, 40 μg / ml of antibody was added to each well for 30 minutes. The supernatant was centrifuged, and the cells were detached from their support using mechanical force (without trypsin) and centrifuged again. After washing with PBS, the cells were fixed at 4°C for 1 hour in 2% glutaraldehyde in 0.1 mol / L sodium phosphate buffer, pH 7.4. Fixation was continued with 1% OsO4 for 15 minutes. Dehydration was performed in ethyl alcohol baths using 25%, 50%, 75%, and 100% ethyl alcohol for 15 minutes in each bath. Finally, the cells were placed on metal stubs and analyzed by scanning electron microscopy.

[0223] As shown in Figure 1, pore formation in the membranes of IMR5 (Panel B), LAN-1 (Panel D), and DUASOII (Panel F) cells is induced by 30 minutes of mAb8B6 treatment. Cells, pore size, and diameter were determined for the three cell types. As shown in Figure 2A, the diameter of the pores induced by mAb8B6 in IMR5 cells was approximately 1.914 μm, and their surface area was approximately 2.877 μm. 2 In this case, the pores occupy at least 4% of the total surface area of ​​the cell. The results obtained for LAN-1 and DUASOII are quite similar. As shown in Figures 2B and 2C, the diameter of the pores induced by mAb8B6 in LAN-1 is approximately 1.760 μm, and its surface area is approximately 2.433 μm. 2 In this case, the pores occupy at least 0.7% of the total surface area of ​​the cell. As shown in Figures 2D and 2E, the diameter of the pores induced by mAb8B6 in DUASOII is approximately 1.459 μm, and its surface area is approximately 1.672 μm. 2 In this case, the pores occupy at least 2.8% of the cell's total surface area.

[0224] Furthermore, the inventors confirmed that lower antibody concentrations induce increased permeability of propidium iodide.

[0225] 2. Treatment with isotretinoin, topotecan, or doxorubicin does not affect OAcGD2 expression on neuroblastoma cell lines. The inventors previously reported the expression of OAcGD2 in neuroblastoma cell lines (Alvarez-Rueda et al., PLos One 2011 6:e25220). Previous studies have shown that GD2 expression—a precursor of OAcGD2—can be altered in neuroblastoma cells upon exposure to retinoic acid (Rebhan et al., Neuroreport. 1994 Apr 14;5(8):941~4 and Hettmer et al., Br J Cancer. 2004 Jul 19;91(2):389~397). Therefore, the inventors investigated whether exposure to anticancer drugs affects mAb8B6 binding levels. To this end, the inventors treated the examined mouse and human neuroblastoma cell lines with each single drug for 48 hours before examining OAcGD2 expression by flow cytometry analysis. The concentrations of the drugs used in these experiments are shown in Table 1.

[0226] Table 1: Drug concentrations used in the treatment of neuroblastoma cells [Table 1]

[0227] The expression of OAcGD2 on the cell surface of neuroblastoma cell lines was analyzed and evaluated by indirect immunofluorescence assay measured by flow cytometry. The inventors evaluated 5 × 10⁻⁶ cells. 5Cells were incubated in 96-well microplates with mAb8B6 (Cerato et al., Hybridoma 1997, 16:307-316) or mAb7H2 (using a mouse monoclonal antibody against O-acetyl GD3 as a negative control antibody) at 10 μg / ml in PBS 1%-BSA for 60 minutes at 4°C. Antibody binding was analyzed after a 60-minute reaction at 4°C using the fluorescein-isothiocyanate conjugate F(ab')2 fragment of goat anti-mouse IgG (VH+VL) as a secondary antibody (Jackson, Immunoresearch, Soham, UK). Cell fluorescence was analyzed using a FACSCalibur® flow cytometer (BD Biosciences, San Jose, CA, USA) and Cell Quest® Pro software (BD Biosciences). The relative fluorescence intensity of 10,000 cells was recorded as a single-parameter histogram (logarithmic scale, 1024 channels, and 40), and the mean fluorescence intensity (MFI) was calculated for each histogram. The results were expressed as an MFI ratio, calculated by dividing the flow cytometry MFI value of cells stained with antigen-specific mAbs by the MFI value of the same cells stained with the negative control mAb 7H2. This method allows for the comparison of multiple test samples within and between different groups.

[0228] As shown in Table 2, the level of mAb8B6 binding on NXS2 cells remained unchanged after 48 hours of incubation with isotretinoin, topotecan, or doxorubicin compared to untreated cells.

[0229] The inventors also found that mAb8B6 binding levels on IMR5, LAN-1, and LAN-5 cells showed little to no change 48 hours after drug incubation (Table 2).

[0230] Table 2: Expression levels of OAcGD2 in neuroblastoma cell lines after 48 hours of anticancer drug exposure. a [Table 2] a The geometric mean fluorescence intensity (MFI) of tumor cells stained with anti-OAcGD2mAb8B6 was normalized to the MFI of tumor cells stained with mAb7H2 control antibody. Data are presented as mean ± SD (n=3). b Untreated cells. C Table 1 shows the concentrations of anticancer drugs used in these experiments.

[0231] 3. Anti-OAcGD2mAb8B6 shows synergistic effects with chemotherapeutic agents in neuroblastoma cell lines. To investigate whether anti-OAcGD2mAb8B6 can enhance chemotherapy, we then characterized the effect of mAb8B6 on tumor cell survival in combination with isotretinoin, topothecan, and doxorubicin in four different neuroblastoma cell lines.

[0232] The antiproliferative activity of anticancer drugs was evaluated using the MTT Cell Proliferation Assay Kit (Roche Diagnostic, Indianapolis, USA). The MTT assay is based on the reduction of yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) by metabolically active cells that form purple formazan crystals. Purple formazan was solubilized with a washing agent and quantified spectrophotometrically at 570 nm.

[0233] 96 wells of cells in the logarithmic phase of proliferation (5 × 10 for LAN-1 and NXS2 cells) 3 , 1 × 10 in the case of IMR5 and LAN-5 cells 4Cells were seeded in 100 μl of complete medium in a treatment plate. Cells were allowed to adhere during overnight incubation before treatment with the test drug. The test drug was serially diluted in complete medium and added to each well by volume 50 / 65 μl, for a total final volume of 165 μl / well. An equal volume of medium was added to the control wells. Three conditions were used: anticancer drug alone, mAb8B6 alone, and mAb8B6 + anticancer drug. Cells were exposed to the test drug for 48 hours. After exposure to the test drug, 15 μl of MTT reagent was added to each well. The plate was returned to the incubator for 4 hours. After the incubation period, the washing reagent (100 μl) supplied from the kit was added to all wells. The plate was wrapped in plastic wrap to prevent evaporation and left overnight at room temperature in the dark. The following day, the absorbance at 570 nm was measured using an iMark® Microplate Absorbance Reader (BioRad). The analysis was performed in sets of four, and the experiment was repeated three times.

[0234] Absorbance values ​​were converted to percentages of the control and plotted against the test drug concentration for maximum half-effective concentration (EC50) calculation using CompuSyn® software (ComboSyn, Paramus, NJ, USA). CompuSyn® is PC computer software developed by Chou and Martin that can be used for dose-effect analysis of single drugs using the median effect equation and multidrug mixtures using both the median effect equation and the combination index equation (2005, CompuSyn for Drug Combinations: PC Software and User's Guide: A Computer Program for Quantitation of Synergism and Antagonism in Drug Combinations, and the Determination of IC50 and ED50 and LD50 Values. ComboSyn, Paramus, NJ). The median effect equation used by CompuSyn® software is derived from the principle of the law of mass action in equilibrium steady state. 50The term " " refers to the concentration of an antibody that induces a response midway between the drug and the maximum after the specified exposure time. The percent value of the control was calculated by dividing the absorbance value of each test well by the no-drug control average and multiplying by 100.

[0235] Combined data were analyzed using CompuSyn® software to calculate combination index values and evaluate synergism. The fraction affected (Fa) was calculated from the percent of the control using the following equation: 1 - (percent control / 100), where 1 corresponds to 100% effect and 0 corresponds to no effect. The doses, fractions affected, and molar ratios of the compounds tested in combination were entered into CompuSyn® software to evaluate the presence of synergism. CompuSyn® assigns a combination index value that evaluates the level of compound that inhibits 50% of cell growth. CI values less than 1 indicate the presence of synergism, CI values greater than 1 indicate antagonism, and a CI50 value equal to 1 indicates an additive effect. See Chou, PHARMACOL. REV., 58(3):621-81 (2006).

[0236] NXS2 cells were grown in DMEM with 4.5 g / L glucose, 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C in 5% CO2. IMR5, LAN-1, and LAN-5 cells were grown in RPMI 1640 with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C in 5% CO2.

[0237] The inventors found similar observations when testing the neuroblastoma cell lines NXS2, IMR5, LAN-1, and LAN-5. The calculated EC50 values of each test chemotherapeutic agent were significantly decreased (p < 0.05, Table 3) in the combination of two agents (i.e., the anticancer agent and mAb8B6).

[0238] Table 3: Enhanced drug-induced cytotoxicity after incubation with mAb8B6 in neuroblastoma cell lines. a [Table 3] a The EC50 concentration is shown as the average of three independent experiments. b Anti-OAcGD2mAb8B6 was combined with chemotherapy at a concentration of 40 μg / ml.

[0239] Finally, the inventors calculated the median combination index values ​​to characterize the effects of all tested combinations. The inventors found that the median combination index values ​​were significantly less than 1.0 (p<0.05) for all tested combinations, indicating synergistic interactions (see Table 4). The combination of isotretinoin and mAb8B6 had combination index values ​​ranging from 0.33 to 0.81 (NXS2=0.74, IMR5=0.81, LAN-1=0.47, LAN-5=0.33) (Table 4). Topotecan showed a stronger synergistic effect, with combination index values ​​ranging from 0.28 to 0.60 (NXS2=0.60, IMR5=0.50, LAN-1=0.58, LAN-5=0.28) (Table 4). The combination with doxorubicin showed a more potent synergistic effect in the test neuroblastoma cell line, with a combination index value of ≤0.30 (NXS2=0.30, IMR5=0.10, LAN-1=0.05, LAN-5=0.16) (Table 4). These data suggest a more potent antineuroblastoma efficacy of chemotherapy when used in combination with anti-OAcGD2mAb8B6.

[0240] Table 4: Combination Index Values [Table 4] *0.05~0.90 = synergistic effect; 0.9~1.10, additive effect; 1.10~10; antagonistic effect.

[0241] Combinations of anti-OAcGD2mAb8B6 with other anticancer agents were also analyzed in LAN-1 neuroblastoma cell lines. In this experiment, intracellular uptake of 5-FU, cisplatin, and doxorubicin in the presence of mAb8B6 or CTRL-(IgG) was detected by flow cytometry (535 nm laser excitation). The anticancer agents and mAbs were incubated at 37°C for 30 minutes. The positive control (0.05% saponin) showed the highest uptake of chemotherapy in LAN-1 cells.

[0242] As shown in Figure 3, when incubated with mAb8B6, more cells were positive for 5-FU (Panel A), cisplatin (Panel B) 1.52 μM, and doxorubicin (Panel C) 4.7 μM than for the same cells incubated with the anticancer drug alone or with CTRL-(IgG). Furthermore, when incubated with doxorubicin (Panel D) 4.7 μM, more cells were positive than for the same cells incubated with the anticancer drug alone or with CTRL-(IgG) than for the same cells incubated with mAb c8B6.14b and mAb c8B6.15b.

[0243] All of these results clearly demonstrate that anti-OAcGD2mAb8B6 promotes the penetration of anticancer drugs into tumor cells.

[0244] 4. Anti-OAcGD2mAb8B6 shows a synergistic effect with temozolomide (TMZ) in primary glioblastoma cells. To test whether mAb8B6 could enhance chemotherapy, the inventors then characterized the effect of mAb8B6 in combination with temozolomide on tumor cell survival in six different glioblastoma primary cells.

[0245] Tumor specimens were collected from patients with a histological diagnosis of GBM. Tumors were collected at the time of surgical resection and cultured immediately after tumor dissection using gentleMACs® Dissociator (Miltenyi) according to the manufacturer's instructions. All specimen collection and analysis were performed according to procedures approved by the Institutional Review Board, and all patients or their guardians provided written informed consent (Comite de Protection des Personnes Ouest IV, Procedure #DC-2012-1555). Cells were maintained in a limited medium (DMEM / Ham F12 containing 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, B27 adjuvant, N2 adjuvant, 2 μg / mL heparin, 40 ng / mL β-FGF, and 40 ng / mL EGF) in a 5% CO2 and 95% humidity atmosphere to form nerve spheres.

[0246] In the case of glioblastoma cells in the logarithmic phase of proliferation, 1 × 10⁶ wells (AMBMa, DUGAn, DUASOII, GLIO5, GUITh, and HARCl). 4 Cells were seeded in 100 μl of complete medium in a treatment plate. Cells were allowed to adhere during overnight incubation before treatment with the test drug. The test drug was serially diluted in complete medium and added to each well by volume 50 / 65 μl, for a total final volume of 165 μl / well. An equal volume of medium was added to the control wells. Three conditions were used: temozolomide alone, mAb8B6 alone, and mAb8B6 + temozolomide. Cells were exposed to the test drug for 72 hours. After exposure to the test drug, 15 μl of MTT reagent was added to each well. The plate was returned to the incubator for 4 hours. After the incubation period, the washing reagent (100 μl) supplied from the kit was added to all wells. The plate was wrapped in plastic wrap to prevent evaporation and left overnight at room temperature in the dark. The following day, the absorbance at 570 nm was measured using an iMark® Microplate Absorbance Reader (BioRad). The analysis was performed in sets of four, and the experiment was repeated three times.

[0247] Cell viability was measured by MTT assay, and the maximum half-maximal effective concentration (EC50) was calculated as previously disclosed. The results are shown in Table 5 below. The inventors observed similar results when testing glioblastoma primary cells AMBMa, DUGAn, DUASOII, GLIO5, GUITh, and HARCl. The calculated EC50 values for each test chemotherapeutic agent decreased with the combination of two drugs (i.e., temozolomide and mAb8B6).

[0248] Table 5: Enhanced cytotoxicity of chemotherapy drugs after incubation with mAb8B6 in primary gliablastoma cells. a

Table 5

[0249] Finally, the inventors calculated the median combination index value to characterize the effect of the combination of tested TMZ and anti-OAcGD2 mAb8B6. The inventors found that the median combination index value was significantly less than 1.0 (p < 0.05) for all combinations tested, indicating a synergistic interaction (see Table 6). The combination of TMZ and mAb8B6 had a combination index value of 0.27 - 0.66 (GUITh = 0.66, DUGAn = 0.58, AMBMa = 0.27, HARCI = 0.30, GLIO5 = 0.41, and DUASOII = 0.65) (Table 6). These data suggest a more potent anti-glioblastoma efficacy of TMZ when used together with anti-OAcGD2 mAb8B6.

[0250] Table 6: Combination index values ​​in primary gliablastoma cells

Table 6

[0251] 5. Anti-OAcGD2mAb8B6 shows synergistic effects with chemotherapy drugs in other types of cancer. To investigate whether anti-OAcGD2mAb8B6 can enhance chemotherapy in cancers other than neuroblastoma and glioblastoma, cell lines describing melanoma (M21 cells), breast cancer (MDA-MB-231 cells overexpressing OAcGD2 ganglioside), small cell lung cancer (H524 cells), glioblastoma (DUGAn cells), and Ewing's sarcoma (TC71 cells) were incubated for 30 minutes with doxorubicin (0.26 μM, except for MDA-MB-231 cells with a doxorubicin concentration of 0.18 μM and DUGAN cells with a doxorubicin concentration of 1 μM) and specific combinations of mAb8B6 or CTRL-(IgG). The number of doxorubicin-positive cells, as previously disclosed in Part 3 of the Examples, was counted using flow cytometry.

[0252] As shown in Figure 4, anti-OAcGD2mAb8B6 significantly increases the proportion of doxorubicin-positive cells in all types of cancer. More specifically, a six-fold increase in doxorubicin-positive cells was observed in the M21 melanoma cell lineage (Panel A), a more than twofold increase in doxorubicin-positive cells was observed in the TC71 Ewing sarcoma cell lineage (Panel D), a nearly twofold increase in doxorubicin-positive cells was observed in the MDA-MB-231 breast cancer cell lineage (Panel B), a 1.5-fold increase in doxorubicin-positive cells was observed in the H524 small cell lung cancer cell lineage (Panel C), and a 1.16-fold increase in doxorubicin-positive cells was observed in DUGAn glioblastoma cells (Panel E).

[0253] In summary, these results demonstrate that anti-OAcGD2mAb8B6 promotes the penetration of anticancer drugs in several types of cancer, particularly cancers with cells expressing O-acetylated GD2 gangliosides.

[0254] 6. Anti-OAcGD2 8B6mAb enhances the antitumor activity of chemotherapeutic agents in a mouse model of neuroblastoma liver metastasis. 6.1 - Mouse Tumor Model The anti-neuroblastoma efficacy of anti-OAcGD2mAb8B6 and chemotherapy treatment was investigated in a mouse NXS2 neuroblastoma experimental liver metastasis model in A / J mice, as previously described by Lode et al. (J Natl Cancer Inst 1997, 89:1586~1594). This study was conducted strictly in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the French Department of Agriculture. The procedure was approved by the Committee on the Ethics of Animal Experiments of the Region Pays de la Loire. Mice were housed at the UTE-UN animal facility (Nantes, France). Female and male A / J mice (6-8 weeks old) were obtained from Harlan Laboratories (Gannat, France). The inventors used 2.5 × 10⁻⁶ mice. 5 Individual tumor cells were inoculated into PBS via the tail vein. The inventors divided the mice into nine groups of 10 mice each: 1) vehicle treatment group; 2) control antibody treatment group; 3) anti-OAcGD2mAb8B6 treatment group; 4) isotretinoin treatment group; 5) topotecan treatment group; 6) doxorubicin treatment group; 7) isotretinoin + mAb8B6 treatment group; 8) topotecan + mAb8B6 treatment group; and 9) doxorubicin + mAb8B6 treatment group. Anti-OAcGD2mAb8B6 treatment was initiated 3 days after tumor cell injection. Mice were administered 25 μg of mAb8B6 twice weekly for 3 consecutive weeks by intravenous injection (days 3, 7, 10, 14, 17 and 21; total dose = 150 μg). Isotretinoin was diluted with Ora-Plus(C) and administered orally at a dose of 10 mg / kg daily for two consecutive weeks (days 10-14 and 17-21). Topotecan diluted with PBS was administered intraperitoneally at a dose of 0.36 mg / kg daily for one week (days 10-14). Doxorubicin was administered intraperitoneally at a dose of 1 mg / kg daily for two weeks (days 10-14 and 17-21). Mice were sacrificed 28 days after inoculation, and antitumor efficiency was evaluated by liver weight and the number of liver metastases in the new specimen.

[0255] 6.2-ResultsTo extend our in vitro observations, we then evaluated the potential therapeutic effect of the mAb8B6 / chemotherapy combination in vivo. We performed in vivo testing using the mouse NSX2 neuroblastoma experimental liver metastasis model disclosed by Alvarez-Rueda et al. (ALVAREZ-RUEDA et al., PLoS One 2011, vol.6(9), p:e25220). To minimize associated side effects, the anticancer drug was administered at low doses previously reported in the literature. Three days after intravenous inoculation of NSX2 tumor cells, 10 mice were treated with either a single agent or the aforementioned mAb8B6 and anticancer drug combination as described above. Twenty-eight days after tumor cell inoculation, we measured the number of liver metastases and liver weight after euthanasia of the mice. The dose of mAb8B6 used by the inventors in this study (cumulative dose = 150 μg) resulted in a significant reduction in NXS2 liver metastases, as indicated by liver weight compared to vehicle-treated mice. The mean liver weight in the mAb8B6-treated group was 1.5 ± 0.15 g compared to 2.5 ± 0.18 g in the vehicle-treated group (p < 0.05, Figure 5). Treatment with an equivalent amount of nonspecific antibody was completely ineffective (mean liver weight = 2.5 ± 0.18 g compared to vehicle-treated mice (data not shown), p > 0.05), demonstrating the specificity of anti-OAcGD2 mAb8B6 therapy. Antibody 8B6 worked in conjunction with isotretinoin (10 mg / kg, oral, 5 times a week for 2 weeks) to induce significant sensitization of NXS2 metastatic proliferation to isotretinoin inhibition (p < 0.05, Figure 5 Panel A). The mean liver weight in the isotretinoin + mAb8B6 treatment group was 1.0 ± 0.06 g, compared to 2.0 ± 0.24 g in the isotretinoin treatment group. The combination of topoisomerase I topotecan (0.36 mg / kg, intraperitoneal, 5 times a week for 1 week) + mAb8B6 also significantly reduced liver weight (0.9 ± 0.03 g) compared to topotecan (1.22 ± 0.09 g) or mAb8B6 alone (p< 0.05, Figure 5 Panel B). The combination of the anthracycline antibiotic doxorubicin (1 mg / kg, intraperitoneal, 5 times a week for 2 weeks) also significantly reduced liver weight (1.33 ± 0.12 g) compared to treatment with doxorubicin (1.75 ± 0.09 g) or mAb8B6 alone (p< 0.05, Figure 5 Panel C).The combination of mAb8B6 with isotretinoin or topotecan produced the strongest therapeutic effect among the three combination dosing regimens tested (Figure 5).

[0256] Weight loss is used as a sensitive marker for health monitoring. Therefore, we performed a parallel analysis of body weight over the treatment period. We observed no weight loss (Figure 6), suggesting no treatment-related toxicity in mice treated with mAb8B6, isotretinoin, isotretinoin + mAb8B6, topotecan, or topotecan + mAb8B6. Mice administered with doxorubicin or doxorubicin + mAb8B6 showed weight loss at day 17 compared to mice administered with mAb8B6 alone. However, the weight difference between the two doxorubicin-treated groups compared to the non-doxorubicin group was not significant during the treatment period (p>0.05). These observations suggest that the combination of anticancer agents + mAb8B6 demonstrates potent antitumor efficacy in vivo compared to either agent alone, without detectable toxicity.

[0257] 7. Anti-OAcGD2 8B6mAb restores the anti-cancer effect of chemotherapy drugs in a refractory glioblastoma model. 7.1 Materials and Methods 7.1.1 - Pharmacologically active substances Anti-OAcGD2mAb8B6 and isotype control antibodies (CTRL) were obtained as previously described. Temozolomide (TMZ) was purchased from Interchim (Montluçon, France). TMZ was reconstituted with DMSO, and a fixed amount was stored at -20°C. DMSO in the same final proportion as the TMZ solution served as a vehicle control in all studies.

[0258] 7.1.2-Cell culture Patient-derived glioblastoma GBM-10 cells were maintained as neurospheres in DMEM / HamF12 supplemented with L-glutamine, B27, N2 supplement, and heparin (2 μg / ml) containing 1% penicillin and streptomycin, and additional growth factors β-FGF (40 ng / ml) and EGF (40 ng / ml) were added immediately, respectively. Culture reagents were obtained from Gibco Life Technologies (Waltham, MA). All cell types were maintained at early passage and examined for mycoplasma by PCR according to the conventional method.

[0259] 7.1.3 - O-acetyl-GD2 expression in GBM10 cells Analysis of OAcGD2 expression in GBM cells was performed by indirect fluorescent antibody method measured by flow cytometry. Cells were washed with cold PBS, fixed with 4% PFA (Electron Microscopy Sciences, Hatfield, PA) for 10 minutes at 4°C, and then incubated with mAb8B6 (10 μg / ml) for 45 minutes. Antibody 8B6 binding was detected by incubating with fluorescein isothiocyanate-labeled F(ab’)2 fragment of goat anti-mouse IgG (Jackson Immunoresearch, Soham, UK) for 60 minutes at 4°C. Another experiment was performed using control IgG. Cell fluorescence was analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Flowjo LLC, Oregon, OR, USA). Results were expressed as MFI ratio.

[0260] 7.1.4 - Limiting dilution analysis For the limiting dilution assay, GBM tumors were dissociated and the isolated cells were initially concentrated at 10 3Cells were seeded at a concentration of cells / mL and then serially diluted in 96-well plates. The cells were cultured for 15 days, after which the percentage of wells without nerve globules was calculated for each cell culture density. The data were analyzed using ELDA software (ELDA, Linz, Austria) to quantify the frequency of glioblastoma stem cells (GSCs) in the samples and the effects of various treatments.

[0261] 7.1.4 - Glioblastoma xenograft mouse model NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice purchased from Charles River Laboratories (Wilmington, MA) were reared in SPF (Specific Pathogen Free) conditions at the animal facility of the University of Nantes (UTE, SFR F.Bonamy) and used at 6-12 weeks of age according to the facility guidelines (Agreement#00186.02; Regional ethics committee of the Pays de la Loire, France). GBM-10 cells (1 × 10⁶ cells in 100 μl Matrigel) 6 (Corning, NY, USA) was administered subcutaneously on day 0. TMZ was administered intraperitoneally at a single dose of 0.05 mg / mouse on days 12, 22, and 32.

[0262] 7.2-Results- TMZ refractory GBM-10 cells (GBM-10TMZ R O-acetyl-GD2 was isolated from relapsed GBM-10 cells after TMZ chemotherapy (Figure 7, Panel A). O-acetyl-GD2 expression levels were measured by flow cytometry analysis as described in the Materials & Methods section. The results show that O-acetyl-GD2 expression persists after TMZ chemotherapy compared to GBM-10 cells isolated from untreated mice (Figure 7, Panel B).

[0263] Next, the TMZ refractory level was determined by limiting dilution assay and expressed as the frequency of glioblastoma cancer stem cells in tumor xenografts. The results showed that stem cell frequency increased with TMZ chemotherapy (Figure 7 Panel C & D). In fact, in untreated mice, both TMZ and mAb8B6 exposure reduced stem cell survival (Figure 7 Panel C). More importantly, the effect induced by the combination of the two drugs (TMZ + 8B6) was significantly higher than that of the two drugs used individually as monotherapies.

[0264] TMZ chemotherapy was extremely inefficient in inhibiting stem cell survival in GBM-10 cells isolated from relapsed GBM-10 xenografts (Figure 7, Panel D). Surprisingly, a combination of the two drugs (TMZ + 8B6) was able to reduce GSC survival.

Claims

1. A composition for use in delivering an anticancer drug into cells expressing OAcGD2 ganglioside, (i) At least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, (ii) comprising at least one multimeric antibody that recognizes the OAcGD2 ganglioside, At least one of the multimeric antibodies that recognizes the OAcGD2 ganglioside a) In amino acid sequence number 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. It contains a heavy chain variable region (VH) where the amino acid at position 115 is V. The composition wherein the multimeric antibody that recognizes the OAcGD2 ganglioside generates nonselective pores within the cell membrane.

2. A composition for use in delivering an anticancer agent to cells expressing OAcGD2 ganglioside, comprising (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons and (ii) at least one multimeric antibody that recognizes OAcGD2 ganglioside, At least one of the multimeric antibodies that recognizes the OAcGD2 ganglioside a) In amino acid sequence number 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. It contains a heavy chain variable region (VH) where the amino acid at position 115 is V. The composition wherein the at least one multimeric antibody that recognizes the OAcGD2 ganglioside enhances the uptake of the at least one anticancer agent by tumor cells.

3. A composition for use in the selective delivery of at least one anticancer agent having a molecular weight of 100 to 200,000 daltons into cells expressing OAcGD2 ganglioside in order to treat and / or prevent cancer expressing OAcGD2 ganglioside, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, (ii) comprising at least one multimeric antibody that recognizes the OAcGD2 ganglioside, a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is V. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at the 24th position is T. The amino acid at position 26 is E. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The composition comprising a heavy chain variable region (VH) in which the amino acid at position 115 is V.

4. The at least one multimeric antibody that recognizes the OAcGD2 ganglioside, a) A light chain variable region (VL) polypeptide having the amino acid sequence of SEQ ID NO: 39, and / or b) A heavy chain variable region (VH) having an amino acid sequence selected from the group including or consisting of SEQ ID NOs: 48, 49, 50, and 51. The composition according to claim 2 or 3, comprising:

5. The composition according to claim 2 or 3, wherein the functional fragment of the at least one multimeric antibody that recognizes the OAcGD2 ganglioside is a single-chain portion of a chimeric antigen receptor (CAR).

6. The at least one multimeric antibody that recognizes the OAcGD2 ganglioside, a) Light chain variable region (VL) polypeptides having amino acid sequences selected from the group including or consisting of SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 39, and / or b) The composition according to claim 2 or 3, comprising a heavy chain variable region (VH) having an amino acid sequence selected from the group including or consisting of SEQ ID NOs: 41, 42, 44, 45, 46, 47, 48, 49, 50, and 51.

7. The at least one multimeric antibody that recognizes the OAcGD2 ganglioside, a) A heavy chain comprising three heavy chain complementarity determining regions (CDRs) of amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and a heavy chain framework sequence derived from an immunoglobulin heavy chain, and b) A light chain comprising three light chain complementarity determining regions (CDRs) of amino acid sequence SEQ ID NOs. 19, SEQ ID NOs. 20, and SEQ ID NOs. 21, and a light chain framework sequence derived from an immunoglobulin light chain. The composition according to claim 2 or 3, comprising:

8. The composition according to claim 2 or 3, wherein the at least one multimeric antibody that recognizes the OAcGD2 ganglioside is an immune complex.

9. The composition according to claim 2 or 3, wherein the at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons is selected from the group comprising or consisting of nucleic acids such as alkylating agents, antimetabolites, antitumor antibodies, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors, corticosteroids, hormones or hormone-like drugs, cytokines, nucleoside analogs, double-stranded synthetic short RNA molecules (miRNA), or synthetic DNA / RNA-like oligonucleotides (ASOs).

10. The composition according to claim 2 or 3, wherein at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons cannot pass through the cell membrane of cancer cells on its own.

11. The composition according to claim 3, wherein the cancer expressing the OAcGD2 ganglioside is selected from the group including or consisting of neuroblastoma, glioma, retinoblastoma, Ewing family tumors, sarcomas (i.e., rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal cell carcinoma, head and neck cancer and hematological cancers (i.e., leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and myeloma).

12. The composition according to claim 11, wherein the at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons is selected from the group comprising or consisting of cyclophosphamide, doxorubicin, topotecan, irinotecan, temozolomide (TMZ), retinoic acid (RA), 5-fluorouracil (5-FU), fludarabine, carboplatin, and cisplatin.

13. The composition according to claim 11, wherein the at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons is temozolomide, topotecan, irinotecan, fludarabine, cyclophosphamide, or a mixture thereof, and the cancer expressing the OAcGD2 ganglioside is neuroblastoma.

14. A composition for use in enhancing the efficacy of treating cancers expressing OAcGD2 ganglioside, The aforementioned treatment includes an anticancer drug, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside, and optionally (iii) a pharmaceutically acceptable carrier. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The composition comprising a heavy chain variable region (VH) in which the amino acid at position 115 is V.

15. A composition for use in enhancing sensitivity to anticancer drugs in patients with cancer expressing OAcGD2 ganglioside, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside, and optionally (iii) a pharmaceutically acceptable carrier. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The composition comprising a heavy chain variable region (VH) in which the amino acid at position 115 is V.

16. A composition for use in preventing or delaying the development of anticancer drug-resistant cancer in patients with cancer expressing OAcGD2 ganglioside, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside, and optionally (iii) a pharmaceutically acceptable carrier. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The composition comprising a heavy chain variable region (VH) in which the amino acid at position 115 is V.

17. A composition for use in cells expressing the O-acetylated form of GD2 ganglioside to enhance the intracellular uptake of anticancer drugs with a molecular weight of 100 daltons to 200,000 daltons, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, and (ii) a multimeric antibody that recognizes OAcGD2 ganglioside. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. In a light chain variable region (VL) polypeptide where the amino acid at position 105 is Q, and in amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The composition comprising a heavy chain variable region (VH) in which the amino acid at position 115 is V.

18. A composition for use in treating refractory or recurrent cancer in patients with cancer expressing OAcGD2 ganglioside, The composition comprises (i) at least one anticancer agent having a molecular weight of 100 daltons to 200,000 daltons, and (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. It contains a heavy chain variable region (VH) where the amino acid at position 115 is V. The composition wherein the cancer is refractory to or subsequently recurs in response to a first-line cancer treatment comprising one or more anticancer agents selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, tyrosine kinase inhibitors, and nucleoside analogs.

19. A kit for the treatment of cancer expressing OAcGD2 ganglioside, The kit comprises (i) at least one anticancer agent having a molecular weight of 100 to 200,000 daltons, (ii) at least one multimeric antibody that recognizes the OAcGD2 ganglioside, and optionally (iii) a pharmaceutically acceptable carrier. a) In amino acid sequence SEQ ID NO: 3, The first amino acid is D. The amino acid at the 9th position is L. The 10th amino acid is S, The amino acid at the 12th position is P. The 13th amino acid is V. The amino acid at the 14th position is T. The amino acid at the 15th position is P. The amino acid at the 17th position is E. The amino acid at the 18th position is P. The amino acid at the 19th position is A. The 20th amino acid is S, The amino acid at the 21st position is I. The amino acid at position 22 is S, The amino acid at position 25 is S, The amino acid at the 29th position is L. The amino acid at the 30th position is L. The 32nd amino acid is N. The 33rd amino acid is N, The amino acid at position 34 is G. The 35th amino acid is N, The amino acid at position 36 is T. The amino acid at position 37 is F. The 39th amino acid is H, The amino acid at position 41 is Y, The amino acid at position 42 is L. The amino acid at position 44 is K. The amino acid at position 47 is Q. The amino acid at position 48 is S, The amino acid at position 50 is Q. The amino acid at position 51 is L. The amino acid at position 55 is K. The amino acid at position 56 is V. The amino acid at position 58 is N, The amino acid at position 60 is L. The amino acid at position 61 is S, The amino acid at position 63 is V. The amino acid at position 65 is D. The amino acid at position 75 is D. The amino acid at position 79 is K. The amino acid at position 81 is S, The amino acid at position 82 is R. The amino acid at position 83 is V. The amino acid at position 84 is E. The amino acid at position 85 is A. The amino acid at position 88 is V. The amino acid at position 89 is G. The amino acid at position 90 is V. The amino acid at position 92 is either F or Y. The amino acid at position 94 is either S or M. The amino acid at position 96 is S, The amino acid at position 97 is T. The amino acid at position 98 is H, The amino acid at position 99 is I. The amino acid at position 100 is P. A light chain variable region (VL) polypeptide in which the amino acid at position 105 is Q, and b) In amino acid sequence number 2, The first amino acid is E. The fifth amino acid is V. The 13th amino acid is Q. The amino acid at the 16th position is R. The amino acid at the 23rd position is T. The amino acid at position 24 is either T or A. The amino acid at position 26 is either E or G. The amino acid at position 30 is either T or G. The amino acid at position 32 is Y, The amino acid at position 35 is T. The amino acid at position 48 is L. The amino acid at position 49 is G. The amino acid at position 50 is F. The amino acid at position 51 is I. The amino acid at position 54 is R. The amino acid at position 55 is A. The amino acid at position 57 is G. The amino acid at position 58 is Y, The amino acid at position 59 is T. The amino acid at position 60 is T. The amino acid at position 61 is E. The 63rd amino acid is N. The amino acid at position 64 is P. The amino acid at position 76 is N. The amino acid at position 77 is S, The amino acid at position 79 is S, The amino acid at position 80 is I. The amino acid at position 81 is L. The amino acid at position 89 is K. The amino acid at position 90 is T. The amino acid at position 95 is V. The amino acid at position 99 is A. The amino acid at position 114 is L. The kit comprises a heavy chain variable region (VH) in which the amino acid at position 115 is V.

Images