Combined treatments of ewing sarcoma with dinutuximab, an anti GD2 antibody and chemotherapy

Abstract

The present disclosure relates to a combined chemo-immunotherapy method for treating GD2 positive cancer, such as relapsed or high- risk metastatic Ewing sarcoma. In particular, the combination therapy comprises the administration of an anti-GD2 antibody alongside a chemotherapy backbone.

Description

[0001] COMBINED TREATMENTS

[0002] FIELD

[0003] The present invention relates to a combined chemo-immunotherapy method for treating GD2 positive cancer, such as relapsed or high-risk metastatic Ewing sarcoma. In particular, the combination therapy comprises the administration of an anti-GD2 antibody alongside a chemotherapy backbone.

[0004] BACKGROUND

[0005] Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, which predominantly affects children, adolescents, and young adults. As a proportion of cases, ES is the second most prevalent primary malignant bone tumour in young patient groups. ES typically develops in the diaphysis (long bone) but can also arise from the metadiaphysis and metaphysis. The histopathology of ES is well understood with tumours showing a characteristic "small round cell" typing. Typically, ES cells are undifferentiated and rich in glycogen.

[0006] The molecular basis for ES is varied, but the primary initiating mutation has been attributed to the fusion of EWSR1-ETS genes by a specific translocation event. A study using whole genome sequencing revealed that the somatic mutation rate in Ewing sarcoma is low (Tirode, F., et al. Cancer Discov. 2014 Nov;4(ll): 1342-53). The most identified somatic mutations were found in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Tumours harbouring mutations in STAG2 and TP53 were found to have particularly poor patient outcomes. A separate study showed that mutational profiles varied in adults compared to children, with tumours isolated from adults showing significantly more mutations in PIK3R1. By contrast, tumours isolated from children exhibited more mutations in pathways responsible for cell cycle control, DNA repair, and transcriptional regulation (Jagodzinska-Mucha, P., et al Contemp Oncol (Pozn). 2021;25(4):241-248).

[0007] Current first-line treatments for ES are surgery, radiotherapy, and chemotherapy. Chemotherapy treatments are divided into three phases: induction, consolidation, and post-consolidation / maintenance therapy. Combination chemotherapy approaches employing a combination of chemotherapeutic drugs are often a part of first-line treatment. A multi-site clinical trial, iEuroEwing 2012, demonstrated that a combination of Vincristine, Doxorubicin & Cyclophosphamide (VDC) and Ifosfamide and Etoposide (IE) forms a component of an effective chemotherapy regimen (Anderton, J., et al. Trials. 2020 Jan 17;21(1):96). Side effects from the treatment can include an increased risk of infection, cardiotoxicity, and neuropathy.

[0008] Monoclonal antibodies that target specific antigens are becoming increasingly prevalent in oncology. The differences in their mode of action compared to traditional cytotoxic therapies have made them valuable first-line agents as shown with rituximab (leukemia and lymphoma), cetuximab (bowel and head / neck cancer), and trastuzumab (breast and stomach cancer). The disialoganglioside GD2, a glycosphingolipid, has been shown to be expressed on the cell surface of nervous tissue but is rarely expressed in normal tissues. Additionally, has been shown to be expressed on the in several ES cell lines and primary cell cultures. Biopsies from ES tumours have also shown uniform GD2+ expression (Kailayangiri, S., et al. Br J Cancer. 2012 Mar 13;106(6): 1123-33).

[0009] New therapeutic protocols are required for Ewing sarcoma patients, and especially for patients with relapsed or high-risk metastatic disease, particularly those that are newly diagnosed.

[0010] All documents referred to herein are incorporated by reference in their entirety.

[0011] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0012] SUMMARY OF INVENTION

[0013] The present disclosure intends to provide new and inventive protocols that can bring improved outcomes to these patients, such as improved response to treatment, reduced cancer load, reduced number of metastatic sites, better quality of life, or increased survival time. For example, the improved outcomes may include improved event-free survival, improved overall survival, improved overall response rate, improved duration of response, improved complete response rate, improved partial response rate, improved primary tumour volume reduction, improved overall response during and after induction, improved metastatic complete response rate, improved metastatic partial response rate, fewer adverse events or serious adverse events, and / or improved measures of quality of life. Improved outcomes may also include improved progression-free survival. An object of the invention is to provide a treatment for a GD2-positive Ewing Sarcoma in a patient in need thereof.

[0014] In a first aspect, the present invention provides a method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of induction chemotherapy with one or more chemotherapeutic agent(s).

[0015] In a second aspect, the present invention provides a method of treating GD2- positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy in accordance with the first aspect, followed by a combination consolidation chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of consolidation chemotherapy with one or more chemotherapeutic agent(s).

[0016] In a third aspect, the present invention provides a method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof.

[0017] In a fourth aspect, the present invention provides a method of treating relapsed GD2- positive Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma, in a subject in need thereof, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or any combinations thereof.

[0018] In a fifth aspect, the present invention provides a method of treating high-risk metastatic GD2-positive Ewing sarcoma in a subject, especially newly diagnosed high- risk metastatic GD2-positive Ewing sarcoma, wherein at least one metastatic tumoral site is distally located from the lung, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or combinations thereof.

[0019] DETAILED DESCRIPTION

[0020] As described above, the present invention uses an anti-GD2 antibody in combination with a chemotherapy regimen to treat GD2 positive Ewing sarcoma, such as relapsed or high-risk metastatic GD2 positive Ewing sarcoma.

[0021] The molecular properties of anti-GD2 monoclonal antibodies, which have been developed clinically and are derived from 14G2a or 3F8, are described in Sterner et al (2017 Aug 15;20(7): 1681-1691). Antibody 14G2a has been developed as chimeric (murine / human) forms known as chl4.18, in particular, dinutuximab beta (Qarziba®) and dinutuximab (Unituxin®).

[0022] Fragment formats based upon chl4.18 have been reported and include minibodies and scFvs, which after conjugation with MMAE or MMAF can induce cytotoxic and cytostatic effects in GD2+ neuroblastoma cell lines. An independently generated murine antibody 3F8, humanized as naxitamab, has been approved for patients with relapsed or refractory high-risk neuroblastoma (Markham, A. Drugs. 2021 Feb;81(2):291-296). According to Sterner (Cell Reports, supra), humanized 3F8 has an apparent kD of 7.7 nM for binding to GD2, a binding preference for GD2 versus the related glycan structure GT2 of 1500, and a binding preference for GD2 versus the related glycan structure GQ2 of 200, whereas dinutuximab (Unituxin®) has an apparent kD of 60 nM for binding to GD2, a binding preference for GD2 versus GT2 of >5000 and a binding preference for GD2 versus GQ2 of 1000. These differences reflect the different binding regions of the antibodies. Dinutuximab beta (Qarziba®), which is produced in Chinese Hamster Ovary (CHO) cells, and dinutuximab (Unituxin®), which is produced in SP2 / 0 murine hybridoma cells, differ in their glycosylation patterns. Dinutuximab beta has a single N-linked glycosylation site (Asn 293), and mass spectrometry analysis revealed that the heavy chain contains the typical IgG diantennary fucosylated N-glycans with 0, 1, or 2 galactose residues, with a smaller fraction of glycans with sialic acid and oligomannose residues, and no Gal-o-1,3 Gal, typical for IgG expression in CHO cells (European Public Assessment Report (EPAR) of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) for Dinutuximab beta Apeiron (EMA / 263814 / 2017, 23 March 2017)). A humanized version of chl4.18 known as hul4.18K322A is described in W02005 / 070967 and has a point mutation in the Fc region in order to reduce complement-dependent cytotoxicity (CDC) but still maintain antibody-dependent cellular cytotoxicity (ADCC). The reduction in CDC is considered to result in reduced pain associated with the antibody treatment. However, it is shown in US 9,777,068 B2 that the cytolysis capacity of an anti-GD2 antibody as measured by a CDC assay is essential for the anti-tumour effect.

[0023] Dinutuximab beta, also referred to as chl4.18 / CHO or APN311 is licensed in the European Union (EU) subject to additional monitoring as Qarziba®. Dinutuximab beta is indicated for the treatment of high-risk neuroblastoma in patients aged 12 months and above, who have previously received induction chemotherapy and achieved at least a partial response, followed by myeloablative therapy and stem cell transplantation, as well as patients with history of relapsed or refractory neuroblastoma, with or without residual disease. For the authorised uses, Qarziba® is administered at 10 mg / m2 / day as an 8-hour or 24-hour infusion (Dinutuximab Beta Investigator's Brochure. Version 3.0 dated 14 May 2019). Dinutuximab beta is also authorised as a medicinal product in Australia and Israel. Various patents cover methods of using Dinutuximab beta, particularly US 9,777,068 B2 (see also WO 2013 / 189554 Al) which discloses a continuous intravenous infusion regimen for the treatment of GD2 positive cancer with reduced side-effect of pain; and US 9,840,566 B2 and US 10,294,305 B2 which disclose treatment regimens for GD2 positive cancer or neuroblastoma respectively, in which IL-2 is not administered in the same treatment cycle and / or overall treatment period.

[0024] As discussed above, there is an unmet need for improved treatments for GD2-positive Ewing sarcoma in a subject in need thereof. The present invention relies on the use of an anti-GD2 antibody (or a fragment thereof), such as but not limited to dinutuximab or dinutuximab beta, to treat GD2-positive Ewing sarcoma, and / or to prevent, treat, inhibit, or reduce metastasis of a GD2-positive Ewing sarcoma or the recurrence thereof, in patients. These anti-GD2 antibodies (or fragments thereof), such as dinutuximab or dinutuximab beta, are to be used in combination with chemotherapy, such as induction chemotherapy. Without wishing to be bound by theory, it is thought that the treatments of the present disclosure provide improved outcomes to patients, such as improved response to treatment, reduced cancer load, reduced number of metastatic sites, reduced side effects, better quality of life, or increased (such as, event-free) survival and / or remission time. It is also thought that the treatments of the present disclosure will improve progression-free survival.

[0025] Aspects of the disclosure are described in detail below.

[0026] Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0027] It is contemplated that any method, use or treatment described herein can be implemented with respect to any other method, use or treatment described herein. All features disclosed herein in connection with any particular aspect are applicable to each of the other aspects, mutatis mutandis. In particular, all features disclosed herein in connection with the first aspect are applicable to each of the second to fifth aspects, mutatis mutandis. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0028] The term "comprises" or "comprising" will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e. including, among other things). As such, the term "comprises" will include references to the component consisting essentially of (such as consisting of) the relevant features. The term "consists of" or "consisting of" will take its usual meaning in the art, namely indicating that the component includes and is limited to the relevant features.

[0029] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

[0030] Where a numerical range is provided herein for any parameter, it is understood that all numerical subsets of that numerical range, and all the individual integer values contained therein, are provided as part of the invention. As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and includes both events which occur, and events that do not occur.

[0031] It will be appreciated that the disclosure herein of methods of treatment in which one or more substances or compositions are administered to a subject is also a disclosure of (1) the associated substances or compositions for use in those methods; and (2) use of the associated substances or compositions in those methods.

[0032] Chemotherapy

[0033] By "chemotherapy" we include the meaning of a treatment regimen using one or more chemotherapeutic agents. Chemotherapy can be categorised in various phases, such as induction, consolidation, maintenance etc, and the skilled person will appreciate that references herein to chemotherapy typically relate to a phase or portion of a treatment regimen rather than to the chemotherapeutic agent(s) per se.

[0034] By "phase", we refer to a treatment phase as understood in clinical practice. Briefly, a treatment phase refers to a specific stage or portion of an overall treatment strategy. Each phase will comprise one or more treatment cycles of one or more chemotherapeutic agent(s).

[0035] By "induction chemotherapy", we mean an initial phase of first-line cancer treatment which aims to destroy a substantial amount of tumoral cells. The term is also synonymous with "primary therapy", and "primary treatment" as understood in the art. Relatedly, the term "re-induction chemotherapy" describes an induction chemotherapy regimen targeted towards patients with relapsed disease. For avoidance of doubt, "induction chemotherapy" includes "re-induction chemotherapy", but in the context of re-induction chemotherapy, the treatment may not be considered "first-line" for the cancer overall, but it would be considered "first-line" in respect of the relapsed disease. In an embodiment, the induction chemotherapy of any aspect of the invention described herein is a re-induction chemotherapy. It would be understood that a first- line cancer treatment may be administered prior to any local treatment e.g., surgery or radiation therapy.

[0036] By "consolidation chemotherapy", we mean a supplementary treatment phase typically administered after induction chemotherapy which aims to eradicate residual tumoral cells to reduce the likelihood of tumour recurrence. Additional interventions may take place between induction and consolidation chemotherapy, for instance, local interventions including radiation and / or surgery. The use of consolidation chemotherapy can be determined by assessing patient response after induction chemotherapy. This process is known as "staging" in which a patient is assessed according to a suitable method in the art, for instance, CT (computed tomography) scan or direct tissue biopsy.

[0037] By "maintenance chemotherapy", we refer to a further treatment phase which typically takes place after consolidation therapy and is designed to extend the length of remission period. Maintenance chemotherapy can be administered to a patient over a prolonged period (for example months or years). It may be administered in an outpatient setting.

[0038] By "other chemotherapy", we refer to a chemotherapy regimen which cannot be categorised according to either "induction", "consolidation", or "maintenance" chemotherapy. Other chemotherapy may include so-called "salvage chemotherapy" or "rescue chemotherapy", wherein a patient is administered further chemotherapy after standard chemotherapy has proven ineffective or wherein the cancer has not responded to previous interventions. This can include chemotherapy in combination with other known treatments including surgery and / or radiation. In some instances, the term may describe an entire treatment phase.

[0039] Treatment cycles of chemotherapy typically comprise a period of treatment, referred to herein as a "dosing period", in which one or more chemotherapeutic agents are administered to the subject, followed by a period of rest (rest / resting period) within the overall cycle. A treatment cycle begins with the start of administration of one or more chemotherapeutic agents. The duration of the treatment cycle can vary depending on the chemotherapeutic agent(s) used and clinical guidance as to a necessary rest period for the patient to recover. Treatment cycles are typically defined by calendar days. For example, a typical treatment cycle can comprise 14, 21, 28 or 35 days. The length of each treatment cycle is normally the same for each cycle within a given phase. Alternatively, the length of each treatment cycle may vary independently for each cycle within a given phase.

[0040] By "chemotherapeutic agent" we include the meaning of a small molecule drug that induces cytotoxic effects that are useful in the treatment of cancer. Also discussed herein is the administration of antibodies or antigen-binding fragments, which are understood to be "immunotherapy agents". Accordingly, in the context of the present invention, a chemotherapeutic agent does not refer to an immunotherapy agent. The related term "chemotherapy dose" refers to a dose of one or more chemotherapeutic agents in accordance with a chemotherapy regimen.

[0041] The same chemotherapeutic agent(s) can be used in one or more phases of chemotherapy. Hence, a given chemotherapeutic agent is not typically restricted to being useful in a single phase (e.g. only in induction chemotherapy). References herein to e.g. "chemotherapeutic agent(s) of the induction chemotherapy" should not be understood as requiring the chemotherapeutic agent(s) to be solely for induction chemotherapy, as they may also be used in, or drawn from, other phases of chemotherapy. Rather, this phrase refers to chemotherapeutic agent(s) when used in the context of induction chemotherapy.

[0042] Each treatment cycle of chemotherapy comprises one or more chemotherapeutic agent(s), which may be the same or different in separate cycles. For example, a set of chemotherapeutic agents may alternate between successive cycles.

[0043] Methods of Treatment

[0044] In the first aspect of the invention, there is provided a method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of induction chemotherapy with one or more chemotherapeutic agent(s).

[0045] The term "treatment", "treat" or "treating" as used herein, refers to therapeutic (curative) treatment including amelioration. Treatment also includes stopping the disease from developing or slowing further progression of the disease. For example, treatment may include preventing symptoms from worsening, such as reducing the chance of metastasis developing or progressing. The terms "prevention", "prevent" and "preventing" as used herein, may also refer to prophylactic treatment during remission i.e., action taken to prevent disease from relapsing, such as in the context of maintenance chemotherapy.

[0046] The terms "patient", "recipient" and "subject" are used interchangeably. By "a patient", "a subject" or "a recipient" we particularly intend a human patient, subject or recipient. Typically, the patient may be a paediatric patient i.e. < 18 years old, optionally < 12 years old or < 6 years old, at the commencement of the treatment. Alternatively, the patient is an adult i.e. > 18 years old at the commencement of the treatment.

[0047] In the context of this invention, "combination chemotherapy" (such as "combination induction chemotherapy"), including any synonymous phrases, means that both the anti-GD2 antibody and chemotherapeutic agent(s) (such as in the context of an induction chemotherapy phase) are administered in combination as a treatment to the patient, which may be referred to as a combination (chemo)therapy. In particular, for a combination chemotherapy involving an anti-GD2 antibody, we mean that the anti- GD2 antibody is administered either simultaneously or sequentially as defined below.

[0048] In the context of this invention, "simultaneous administration" or "administered simultaneously" is defined by beginning administering the antibody within the dosing period of the one or more chemotherapeutic agents of the respective chemotherapy. For example, the antibody may begin to be administered on the same calendar day as one or more of the chemotherapeutic agent(s) of the respective chemotherapy. For example, the antibody may begin to be administered within one hour of administering one or more chemotherapeutic agent(s) of the respective chemotherapy.

[0049] In the context of this invention, "sequential administration" or "administered sequentially" is defined as administering the antibody after the dosing period of the one or more chemotherapeutic agent(s) of the respective chemotherapy. This means that the antibody is administered fully within the resting period from the chemotherapeutic agent(s). For example, the antibody may begin to be administered one calendar day after the administration of one or more chemotherapeutic agent(s). For example, the antibody may begin to be administered two or more calendar days after the administration of one more chemotherapeutic agent(s). Without limiting the invention, the skilled person will appreciate that in order for the bone marrow to recover, the patient should be given a reasonable period in which neither the chemotherapeutic agents nor antibody are given, and so it would be particularly suitable for administration of the antibody to be given early within the resting period from the chemotherapeutic agent(s).

[0050] In the context of this invention, the method of treatment may typically comprise cycles of 14, 21, 28 or 35 days. In particular, the method of treatment may comprise cycles of 21 days. In some embodiments, the method of treatment may comprise cycles of at least 21 days to 28 days, for example, the treatment may comprise cycles of at least 21, 22, 23, 24, 25, 26, 27 or 28 days. In the context of both simultaneous and sequential administration, the antibody and the one or more chemotherapeutic agents are administered within the same treatment cycle.

[0051] In an embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment dose:

[0052] (a) simultaneously with one or more of the chemotherapeutic agent(s) in the first cycle of the induction chemotherapy;

[0053] (b) as sequential administration with the one or more of the chemotherapeutic agent(s) in the first cycle of the induction chemotherapy;

[0054] (c) simultaneously with one or more of the chemotherapeutic agent(s) in a second or subsequent cycle of the induction chemotherapy, such as in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle;

[0055] (d) simultaneously with one or more of the chemotherapeutic agent(s) in the fifth cycle of the induction chemotherapy;

[0056] (e) as sequential administration with the one or more of the chemotherapeutic agent(s) in the fifth cycle of the induction chemotherapy; or

[0057] (f) as sequential administration with the one or more of the chemotherapeutic agent(s) in the second or subsequent cycle of the induction chemotherapy, such as in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle.

[0058] The first treatment dose of the antibody is the first dose of the antibody administered to a subject in accordance with the methods described herein, or for the phase of combined chemotherapy being discussed.

[0059] In an embodiment of the first aspect of the invention, administered simultaneously means beginning administering the antibody within the dosing period of the induction chemotherapy. For instance, if the one or more chemotherapeutic agent(s) are administered over 5 days, the administration of the antibody may begin on the first, second, third, fourth or fifth day in that dosing period.

[0060] In particular, administered simultaneously may include beginning administering the antibody during the dosing period of one or more of the chemotherapeutic agent(s) of the induction chemotherapy, on the same calendar day. In an embodiment of the first aspect of the invention, administered simultaneously means administering the antibody within the dosing period of the one or more chemotherapeutic agent(s) of the induction chemotherapy contemporaneously to the extent that the antibody begins to be applied within one hour of a dose of one or more of the chemotherapeutic agent(s).

[0061] In an embodiment of the first aspect of the invention, the antibody in the combination induction chemotherapy is administered in a first treatment dose (a) simultaneously with one or more of the chemotherapeutic agent(s) in the first cycle of the induction chemotherapy.

[0062] In another embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment cycle (b) as sequential administration with the one or more chemotherapeutic agent(s) in the first cycle of the induction chemotherapy. This includes that the administration of the first treatment dose of the antibody and the administration of the one or more chemotherapeutic agents do not take place on the same calendar day in the first cycle. As a non-limiting example, one or more chemotherapeutic agent(s) may be given on days 1 to 4 of the first cycle, followed by administration of the antibody beginning on day 5 and ending before the first cycle is finished.

[0063] In an embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment dose (c) simultaneously with one or more of the chemotherapeutic agent(s) in a second or subsequent cycle of the induction chemotherapy. For example, the first treatment dose of the antibody may be simultaneously administered in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle of the induction chemotherapy.

[0064] In an embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment dose (d) simultaneously with one or more of the chemotherapeutic agent(s) in the fifth cycle of the induction therapy.

[0065] In an embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment dose (e) as sequential administration with the one or more of the chemotherapeutic agent(s) in the fifth cycle of the induction chemotherapy This includes that the administration of the first treatment dose of the antibody and the administration of the one or more chemotherapeutic agents in the fifth cycle do not take place on the same calendar day in the fifth cycle. As a non-limiting example, one or more chemotherapeutic agent(s) may be given on days 1 to 4 of the fifth cycle, followed by administration of the antibody beginning on day 5 and ending before the fifth cycle is finished.

[0066] In an embodiment, the antibody in the combination induction chemotherapy is administered in a first treatment dose (f) as sequential administration with the one or more chemotherapeutic agent(s) in the second or subsequent cycle of the induction chemotherapy, such as in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle. Hence, the administration of the first treatment dose of the antibody and the administration of the one or more chemotherapeutic agents in the nth cycle do not take place on the same calendar day in the nth cycle. As a nonlimiting example, one or more chemotherapeutic agent(s) may be given on days 1 to 4 of the nth cycle, followed by administration of the antibody beginning on day 5 and ending before the nth cycle is finished.

[0067] In an embodiment, the antibody in the combination induction chemotherapy is administered:

[0068] (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy;

[0069] (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy;

[0070] (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy; or

[0071] (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy.

[0072] In an embodiment, the antibody in the combination induction chemotherapy is administered (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy. This means that with each dosing period of the one or more chemotherapeutic agent(s), a treatment dose of antibody is administered simultaneously. The context by which the antibody is administered simultaneously may vary independently in each cycle. As a non-limiting example, the dosing period for the one or more chemotherapeutic agent(s) may be four days, and the particular day on which the antibody is administered may differ between cycles, e.g. such that in one cycle it is administered on day 1 of the cycle and in another cycle it is administered on day 2 of the cycle.

[0073] In an embodiment, the antibody in the combination induction chemotherapy is administered (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy. This means that following each dosing period of the one or more chemotherapeutic agent(s), a treatment dose of antibody is administered sequentially. The context by which the antibody is administered sequentially can vary independently in each cycle. For example, in one cycle of induction chemotherapy, the antibody may be administered one calendar day after the administration of the chemotherapeutic agent(s). In another cycle of the induction chemotherapy, the antibody may be administered two or more calendar days after the administration of the chemotherapeutic agent(s).

[0074] In an embodiment, the antibody in the combination induction chemotherapy is administered (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy. As a non-limiting example, if the first dose of antibody is administered in the second treatment cycle of the combination induction chemotherapy which comprises 9 treatment cycles, the antibody would further be administered simultaneously in the third, fourth, fifth, sixth, seventh, eighth, and ninth treatment cycles.

[0075] In an embodiment, the antibody in the combination induction chemotherapy is administered (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy. As a non-limiting example, if the first dose of antibody is administered in the second treatment cycle of the combination induction chemotherapy which comprises 9 treatment cycles, the antibody would further be administered sequentially in the third, fourth, fifth, sixth, seventh, eighth, and ninth treatment cycles.

[0076] Alternatively in another embodiment, the administration of the antibody may be discontinued after one or more treatment cycles, such as after two, three, four, five or more treatment cycles. This can be combined with any embodiment relating to simultaneous or sequential administration of the antibody. The administration of the antibody may be skipped in one or more treatment cycles and then begun again within the same phase of chemotherapy.

[0077] Moreover, the administration of the antibody may be given in alternating cycles, such as in odd numbered or even numbered cycles, whether beginning in the first cycle or in a later cycle within the phase of chemotherapy.

[0078] Without wishing to be bound by theory, when the antibody in the combination induction chemotherapy is administered in a first treatment dose simultaneously with a first treatment dose of one or more chemotherapeutic agent(s) of the induction chemotherapy, there may be a faster response to treatment. Alternatively and without wishing to be bound by theory, when the first dose of the antibody is given later in treatment, there may be reduced side effects, such as a reduced risk of the subject developing a potential tumor lysis syndrome (TLS) or developing cytokine release syndrome (CRS) also known as a cytokine storm. TLS occurs when many cancer cells die within a short period, releasing their contents into the blood and subsequently causing an immunological reaction. Cytokine release syndrome (CRS) happens when your immune system responds to infection or immunotherapy drugs more aggressively than it should. The skilled person would be aware of the clinical indicators associated with TLS and CRS and may choose the alternative dosing specified above if the patient is at risk of TLS and / or CRS. TLS is commonly classified as either laboratory TLS or clinical TLS. Methods for assessing the laboratory and clinical indicators are well established in the art. Further updates to the standard of care would be considered by the skilled person when carrying out the invention. The skilled person would also appreciate that the dosage of the antibody may be selected and / or adjusted to reduce the risk of TLS and / or CRS.

[0079] GD2-positive Ewing sarcoma

[0080] The methods of the invention are for treating a GD2-positive Ewing sarcoma. A GD2- positive Ewing sarcoma is a Ewing sarcoma which expresses or overexpresses GD2. "GD2" may also be referred to as "Ganglioside G2". GD2 is a disialoganglioside which may be expressed on tumours of neuroectodermal origin, including human neuroblastoma and melanoma, and typically has highly restricted expression on normal (i.e. non-cancerous) tissues.

[0081] GD2 surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies which may be able to eradicate high- risk metastatic cells and prevent relapse. It will be appreciated that to establish that GD2 positive Ewing sarcoma is present, a diagnostic test may be required. Suitably, the Ewing sarcoma tumour is characterised to assess its GD2 positivity prior to treatment. For example, a biological sample, such as a sample of a tumour, may be obtained from the subject and assessed through immunohistochemical means, such as wherein an anti-GD2 antibody is used to confirm the presence of the GD2 antigen.

[0082] In some embodiments in accordance with any of the aspects of the invention disclosed herein, prior to the combination chemotherapy (such as the combination induction chemotherapy of the first aspect of the invention), the method of any aspect described herein comprises the step of determining or confirming that the subject has GD2- positive Ewing sarcoma. Optionally, the subject may have at least one confirmed GD2 positive tumour, biopsy or cell sample. Optionally, the step of determining or confirming that the subject has GD2-positive Ewing sarcoma comprises immunostaining, flow-cytometry, RT-PCR, and / or an ELISA. The step of determining or confirming that the subject has GD2-positive Ewing sarcoma may be carried out using in vitro, ex vivo or in vivo techniques, such as immunostaining on tumour biopsies, flow cytometry, RT-PCR, and ELISA. In a related embodiment, the method comprises the step of determining or confirming that the subject has Ewing sarcoma. Suitably, the method may comprise fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), or next-generation sequencing (NGS) assay.

[0083] Kailayangiri et al (Br J Cancer. 2012 Mar 13;106(6): 1123-33) successfully demonstrated that immunofluorescence staining can detect GD2 expression from tissue cross sections originating from Ewing sarcoma tumours. In summary, detection was carried out on 4 pm cross-sections of tumour tissue after fixation in 2% paraformaldehyde for 10 min, then subsequent permeabilization for 3 h in phosphate- buffered saline (PBS) containing 0.075% Tween and 1% bovine serum albumin. Sections were then incubated overnight with FITC-conjugated 14.G2a mAb, diluted 1 : 50 (2 / jg / ml) in PBS supplemented with 0.01% Tween, and counterstained with DAPI. GD2 expression was observed in tissue isolated from patients with localised and metastatic disease. A subsequent study demonstrated that flow cytometry can be used to differentiate between GD2 high expression and GD2 low expression sub-population of Ewing sarcoma (Kailayangiri, S., et al. Mol Then 2019 May 8;27(5):933-946). A further example of immunofluorescence staining is provided by WO 2024 / 235874 which demonstrated the efficacy of a near-infrared fluorescent dye-antibody conjugate to detect and track GD2+ tumours in vivo. Detection of Ganglioside GM2 / GD2 Synthetase mRNA has been shown to be a sensitive biomarker in other GD2-positive cancers such as infrequent Neuroblastoma Cells from bone marrow. Hoon et al (Am J Pathol. 2001 Aug; 159(2) :493-500) identified a suitable primers pair to detect GalNAc-T mRNA: 5'-CCA ACT CAA CAG GCA ACT AC-3' (sense; SEQ ID NO: 20) and 5'-GAT CAT AAC GGA GGA AGG TC-3' (antisense; SEQ ID NO: 21) which yields a cDNA product of 230 bp. The skilled person would recognise that a method suitable for the detection of Ganglioside GM2 / GD2 Synthetase mRNA in infrequent Neuroblastoma Cells could be adapted for use in Ewing sarcoma.

[0084] Enzyme-linked immunosorbent assays (ELISA) have been documented for the purpose of detecting the surface expression of GD2 in cancer cell lines. Soman et al (J Immunol Methods. 2011 Oct 28;373(l-2): 181-91) describes a cell binding ELISA (cbELISA) in which anti-GD2 antibodies chl4.18 and hul4.18IL-2 showed dose-dependent binding to the melanoma cell line M21 / P6. The skilled person would recognise that such a method may be adapted and optimised for use in determining the expression of GD2 in Ewing sarcoma tissue and / or isolated cells.

[0085] Further methods for confirming that the subject has Ewing sarcoma, which are typically GD2-positive, can include methods that detect the causative translocation event. For example, the translocation of t(ll;22)(q24;ql2) which fuses the EWS gene on chromosome 22 with the FLU gene on chromosome 11. Suitable methods for detecting the translocation event can comprise fluorescence in situ hybridization (FISH), realtime polymerase chain reaction (RT-PCR), or next-generation sequencing (NGS) assay. Each method may be performed on biopsy samples in vitro or using ex vivo tissue.

[0086] In an embodiment, the subject has relapsed or high-risk metastatic Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma or newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma.

[0087] In an embodiment, the subject has relapsed Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma. "Relapsed" means a cancer that has come back after a period of improvement (e.g., due to treatment), such as after a period of remission or stable disease. There is no time limit on how long a cancer can be in a period of improvement or remission before a returning cancer is referred to as "relapsed", but typically the period of improvement or remission may be in the order of months, or years. For instance, a patient with relapsed cancer may have already undergone initial treatment for the cancer, resulting in a period of improvement (such as a period with reduced symptoms of cancer, and / or an improved prognosis), stable disease, and / or a period of partial or complete remission, followed by the return of the cancer (relapsed cancer). For the avoidance of doubt, relapse can recur multiple times. The present invention may be used in respect of any of these recurrences, for example, the invention may be used to treat the first, second, third or any subsequent relapse of Ewing sarcoma. Patients with second or later relapses represent the same disease continuum and share similar therapeutic challenges. Remission means that the signs and / or symptoms of the cancer are reduced. Remission can be partial or complete. In a complete remission, all signs and symptoms of cancer may have disappeared, so that the cancer can no longer be detected in the patient. In partial remission, some signs and symptoms of cancer may be reduced and / or have disappeared, but some signs and / or symptoms may remain. A "relapsed" cancer may also be referred to as a "recurrent" cancer.

[0088] Accordingly, in some embodiments, the subject has an initial relapsed Ewing sarcoma. In other embodiments, the subject has a second relapsed Ewing sarcoma. In further embodiments, the subject has a further relapsed sarcoma.

[0089] Optionally, the subject has relapsed 3, 4, 5 or 6 or more months after:

[0090] (i) a remission diagnosis;

[0091] (ii) the completion of the previous therapy regimen; and / or

[0092] (iii) commencement of maintenance therapy.

[0093] Preferably, the subject has relapsed after 6 or more months, which is understood as being "late relapse". "Early relapse" refers to when the subject has relapsed after less than 6 months.

[0094] By "remission diagnosis" we mean that the subject has been diagnosed as being in partial or complete remission. In an embodiment, the subject has relapsed 6 or more months after a complete remission diagnosis. In an embodiment, the subject has relapsed 6 or more months after a partial remission diagnosis. For example, the relapse may be 6, 7, 8, 9, 10, 11, 12, 15, 18 or 24 months or more after a remission diagnosis.

[0095] By "completion of the previous therapy regimen" we mean any treatment that was administered to the subject as an attempt to treat at least one site (primary or metastatic) of Ewing sarcoma has ended.

[0096] By "commencement of the maintenance therapy", we refer to the start of a maintenance therapy following the successful completion of a suitable therapy. This suitable therapy could include a treatment regime of either radiotherapy, surgery, induction chemotherapy, consolidation chemotherapy, other chemotherapy, or combinations thereof. In particular, in the context of the present invention, the maintenance therapy would follow therapy that includes at least one chemotherapy. The time period would be recognisable by the skilled person based upon the subject's treatment history.

[0097] "Metastatic" refers to a cancer that has spread (or is likely to spread) from the primary site to other parts of the body. Such a cancer may be further characterized by the mechanism(s) by which the cancer spreads. For example, a cancer may spread through direct extension into the tissues or structures surrounding the primary site; through the lymphatic system; or by bloodstream infiltration. When tumour cells metastasize, the new tumour may be called a secondary or metastatic tumour, and its cells are typically similar to those in the original or primary tumour. Metastasis is typically a key element in cancer staging systems. For example, in overall stage grouping, metastasis may place a cancer in Stage IV. The possibilities of curative treatment are typically reduced, or may be entirely removed, when a cancer has metastasised.

[0098] In an embodiment, the subject has high-risk metastatic Ewing sarcoma, particularly newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma. In some embodiments, "high-risk metastatic" refers to a Ewing sarcoma wherein the primary site and one or more additional tumoral sites are distally located from the lung. Conversely, a "low-risk metastatic" can refer to a Ewing sarcoma wherein, other than the primary site, one or more additional tumoral sites are predominantly located in the lung. The skilled person would be able to distinguish between different forms of metastatic Ewing sarcoma by referring to clinical guidance. Optionally, the cancer (Ewing sarcoma) in the subject has metastasised from the bone or soft tissue to at least one other site (may be referred to as a metastatic site or secondary site) within the body. Suitably, the subject has at least one metastatic tumoral site that is distally located from the lung. Alternatively, the subject has no metastatic sites in the lung.

[0099] In an embodiment, the subject has low-risk metastatic Ewing sarcoma.

[0100] In an embodiment, the subject has not previously received treatment with an anti-GD2 antibody for the treatment of Ewing sarcoma. Optionally, the subject has not previously been treated with an anti-GD2 antibody for any purpose. In an embodiment, the subject has not been treated with an anti-GD2 antibody for any cancer. In an embodiment of any aspect described herein, the subject may be newly diagnosed with Ewing sarcoma, such as newly diagnosed with relapsed and / or high-risk metastatic Ewing sarcoma or low-risk metastatic Ewing sarcoma. The term "newly diagnosed" is well understood in the art. By "newly diagnosed" we particularly include the meaning that the subject has received a Ewing sarcoma diagnosis (including high- risk metastatic and / or relapsed) but has not commenced any treatment since the diagnosis. This includes subjects who may be diagnosed with Ewing sarcoma for the first time, subjects who may have previously received a Ewing sarcoma diagnosis but since went into full or partial remission (the diagnosis may accordingly be relapsed Ewing sarcoma), and / or subjects who have received a new diagnosis due to, e.g., metastasis of the cancer (the diagnosis may accordingly be high-risk metastatic Ewing sarcoma). Suitably, the subject may have received a new Ewing sarcoma diagnosis (of any type), in the day, week, 1 month, 2 months, 3 months or 4 months before commencing treatment according to a method described herein.

[0101] The methods of the invention can be applied to the treatment of Ewing Sarcoma Family of Tumours (ESFT). ESFT is characterized by the same neural origin as Ewing Sarcoma, similar small round-blue cell morphology and common chromosomal translocations involving the Ewing sarcoma breakpoint region 1 EWSR1) gene. For example, ESFT may include any of Ewing tumour of bone (ETB or Ewing sarcoma of bone), extraosseous Ewing (EOE) tumours, primitive neuroectodermal tumours (PNET or peripheral neuroepithelioma), Askin tumours (PNET of the chest wall), and Ewing-like sarcomas. Ewing Sarcoma and ESFT can be managed with the same complex multimodal treatment approach. Hence, in some embodiments, the treatment described herein may be used to treat ESFT.

[0102] Chemotherapeutic agents

[0103] By "chemotherapeutic agent" we mean an active agent that is used in chemotherapy. As described previously, a chemotherapeutic agent refers to a small molecule drug and excludes biologies and other immunotherapy agents.

[0104] In an embodiment, one chemotherapeutic agent is administered to the subject as part of the induction chemotherapy.

[0105] In some embodiments, one chemotherapeutic agent is included in the combination induction chemotherapy. The one chemotherapeutic agent may be selected from any chemotherapeutic agent referenced below. For example, the single chemotherapeutic agent may be high dose ifosfamide (HD IFOS).

[0106] Suitably, at least one cycle of the induction chemotherapy is administered, such as at least 4 cycles, such as at least 9 cycles. Suitably, from two to ten cycles of the induction chemotherapy are administered to a subject, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles. In one particular embodiment, 4 cycles of the induction chemotherapy are administered to a subject, such as 4 cycles of HD IFOS.

[0107] In an embodiment, at least 2, at least 3, or at least 4 chemotherapeutic agents are administered to the subject as part of the induction chemotherapy. In an embodiment, two chemotherapeutic agents are administered to the subject as part of the induction chemotherapy. In an embodiment, three chemotherapeutic agents are administered to the subject as part of the induction chemotherapy. In an embodiment, four chemotherapeutic agents are administered to the subject as part of the induction chemotherapy.

[0108] In a particular embodiment, five chemotherapeutic agents are administered to the subject as part of the induction chemotherapy.

[0109] In an embodiment, the chemotherapeutic agents administered to the patient are in combinations selected from:

[0110] (i) vincristine, doxorubicin, and cyclophosphamide (VDC);

[0111] (ii) ifosfamide, and etoposide (IE);

[0112] (iii) vincristine, ifosfamide, doxorubicin, and etoposide (VIDE);

[0113] (iv) vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide (VDCIE);

[0114] (v) temozolomide and irinotecan (TEMIRI); and / or

[0115] (vi) topotecan and cyclophosphamide (TOPOCYCLO).

[0116] In some embodiments, one combination of chemotherapeutic agents is administered in each cycle of induction chemotherapy. For example, a combination of temozolomide and irinotecan may be administered in each cycle of induction chemotherapy. As an alternative example, a combination of topotecan and cyclophosphamide (TOPOCYCLO) may be administered in each cycle of induction chemotherapy.

[0117] Suitably, at least one cycle of the combination induction chemotherapy is administered, such as at least two cycles, at least four cycles, at least 6 cycles or up to 12 cycles or more. For example, 6 cycles of the combination induction chemotherapy are administered to a subject. As alternative examples, 7, 8, 9, 10, 11 or 12 cycles of the combination induction chemotherapy are administered to a subject. In particular, 6- 12, such as 6 or 12, cycles of the combination induction chemotherapy may be administered to a subject, wherein the chemotherapeutic agent combination is selected from temozolomide and irinotecan (TEMIRI) or topotecan and cyclophosphamide (TOPOCYCLO).

[0118] In an embodiment, two or more chemotherapeutic agent combinations are administered in alternating cycles of induction chemotherapy. In an embodiment, IE and VC are administered in alternating cycles of induction chemotherapy. In an embodiment, VDC and IE are administered in alternating cycles of induction chemotherapy.

[0119] Suitably, at least two cycles of induction chemotherapy are administered, such as at least 5 cycles, such as at least 9 cycles. Suitably, from two to ten cycles of induction chemotherapy are administered to a subject, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles.

[0120] Suitably, the chemotherapeutic agents of any aspect described herein may be selected from one or more of the following vincristine, ifosfamide, cyclophosphamide, doxorubicin, and etoposide, temozolomide, irinotecan, topotecan and optionally actinomycin D. The skilled person will appreciate how these chemotherapeutic agents can suitably be administered.

[0121] Vincristine (V) is a chemotherapeutic agent that belongs to a group of agents called vinca alkaloids. Vincristine may particularly be administered at a dose of at least 2 mg / m2, optionally by IV infusion. Vincristine may be provided as a solution for injection.

[0122] Ifosfamide (I) may particularly be administered at a dose of at least 1800 mg / m2 / d, optionally by IV infusion, such as Ihr IV infusions over 5 days. Ifosfamide may be provided as a powder for concentrate for solution for infusion.

[0123] High dose ifosfamide (HD IFOS) may be administered at a dose of at least 3 g / m2 / d for 5 days, such that the total dose within a treatment cycle is 15 g / m2. The skilled person would select an appropriate dose in accordance with the local standard of care. In some instances, HD IFOS may be administered with Lenvatinib. Lenvatinib is administered as a capsule taken once daily continuously throughout treatment. Cyclophosphamide (C or CYCLO) may particularly be administered at a dose of at least 1200 mg / m2, optionally by IV infusion, orally (such as by tablet), or IV injection. Cyclophosphamide may be provided as a powder for solution for injection.

[0124] Doxorubicin (D) may particularly be administered at a dose of at least 37.5 mg / m2 / d, optionally for 24 hours, such as by IV infusion for 24 hours. Doxorubicin may be provided as a solution for infusion.

[0125] Etoposide (E) may particularly be administered at a dose of at least 100 mg / m2optionally by IV infusion, such as Ihr IV infusions over 5 days. Etoposide may be administered intravenously, such as by IV infusion, or administered orally, such as by capsule. Etoposide may be provided as a concentrate for solution for infusion.

[0126] Temozolomide (TEM) may particularly be administered at a dose of at least 75 mg / m2optionally by intravenous infusion or orally administration. Temozolomide may be administered intravenously, such as by IV infusion, or administered orally, such as by capsule. Temozolomide may be provided as a 100 mg lyophilized powder in singledose vial for reconstitution or in capsules containing 5 mg, 20 mg, 100 mg, 140 mg, 180 mg, or 250 mg of the agent.

[0127] Topotecan (TOPO) may particularly be administered at a dose of at least 1.5 mg / m2optionally by IV infusion, such as for 30 minutes daily for up to 5 days. Topotecan may be administered intravenously, such as by IV infusion. Topotecan may be provided as a single-use vial containing 4 mg of topotecan free base as a 4 mg / 4 mL (1 mg / ml final concentration). Alternatively, Topotecan may be administered in a 3-weekly (21 day cycle) short infusion schedule of 2 mg / m2 / day for 5 days per cycle.

[0128] Irinotecan (IRI) may particularly be administered at a dose of at least 125 mg / m2intravenous infusion, such as for 90 minutes daily. Irinotecan may be administered intravenously, such as by IV infusion. Irinotecan may be provided as single use vials, containing, 40, 100, or 300 mg of an irinotecan hydrochloride solution.

[0129] Gemcitabine (G) may particularly be administered at a dose of at least 900 mg / m2. Gemcitabine may be administered twice, on day 1 and 8 of a treatment cycle.

[0130] Docetaxel (Do) may particularly be administered at a dose of at least 80 mg / m2. Docetaxel may be administered on day 8 of a treatment cycle. Carboplatin (C) may particularly be administered at a dose of at least 400 mg / m2intravenously, such as by IV infusion. Carboplatin may be administered on day 1 of a treatment cycle.

[0131] In some instances, chemotherapeutic agents may be administered with busulfan or melphalan.

[0132] In some embodiments, the chemotherapeutic agents administered to the patient are in combinations selected from

[0133] (i) Ifosfamide, Carboplatin and Etoposide (ICE) and / or

[0134] (ii) Topotecan and Temozolomide (TOTEM).

[0135] The administration and dosing schedule of the one or more chemotherapeutic agents may be according to the Euro Ewing 2012 trial (EudraCT Number: 2012-002107-17; "International Randomised Controlled Trial for the Treatment of Newly Diagnosed Ewing's Sarcoma Family of Tumours"), the protocol (especially Supplementary Table 1) of which is incorporated herein by reference. The treatment for Ewing's sarcoma in the trial is of three phases: induction chemotherapy, local control (surgery and / or radiotherapy) and consolidation chemotherapy. The Euro Ewing 2012 trial is an international, phase III, open-label, randomised controlled trial. All patients are randomised at trial entry to receive either the current European treatment strategy or the American equivalent. The European strategy consists of VIDE induction chemotherapy and VAI / VAC (risk-adapted) consolidation chemotherapy, whilst the American strategy consists of compressed VDC / IE induction chemotherapy and IE / VC consolidation chemotherapy. The objective of the trial is to compare these two strategies with respect to clinical outcome and toxicity.

[0136] Suitably, methods of any aspect of the invention may incorporate administration of one or more of vincristine, ifosfamide, cyclophosphamide, doxorubicin, etoposide, temozolomide, irinotecan, and topotecan according to the European strategy or American strategy for induction and / or consolidation chemotherapy.

[0137] Suitably, cycles of VIDE may be given at 21 day intervals (+ / - 3 days) and on haematological recovery to absolute neutrophil count (ANC) > 1.0xl09 / L, platelets >80xl09 / L. VIDE may be administered in accordance with Table 1. Table 1 : VIDE induction chemotherapy

[0138] Patients whose surface area (SA) is > 2 m2may have their doses capped and calculated with a SA of 2 m2.

[0139] Suitably, the induction chemotherapy may follow a VDC / IE chemotherapy dosing schedule. Alternating cycles of VDC and IE may be given, such as at 14 day intervals (+ / - 3 days) and optionally on haematological recovery to absolute neutrophil count (ANC) >0.75xl09 / L, platelets >75xlO9 / L. Blood counts may be obtained on day 7 and 14 of the cycle and every Monday, Wednesday, and Friday after day 14, until the criteria for beginning the next cycle are satisfied. VDC / IE may be administered in accordance with Table 2.

[0140] Table 2: VDC / IE induction chemotherapy

[0141] Patients whose SA is > 2m2may have their doses capped and calculated with a SA of

[0142] 2 m2. Alternating cycles of IE and VC may be given at 14 day intervals (+ / - 3 days) and optionally on haematological recovery to ANC >0.75xl09 / L, platelets >75xlO9 / L. Blood counts may be obtained on day 7 and 14 of the cycle and every Monday, Wednesday, and Friday after day 14, until the criteria for beginning the next cycle are satisfied. IE / VC may be administered in accordance with Table 3. Table 3: IE / VC induction chemotherapy

[0143] *MESNA and hydration should be given according to institutional guidelines

[0144] VC

[0145] Patients whose SA is > 2 m2may have their doses capped and calculated with a SA of 2 m2.

[0146] In an embodiment, the induction chemotherapy is according to the VDC / IE, IE / VC or VIDE dosing schedule described herein.

[0147] In an embodiment, the induction chemotherapy comprises alternating cycles in which either VDC or IE are administered.

[0148] Without wishing to be bound by theory, the induction chemotherapy selected for use in the invention may comprise any standard-of-care chemotherapy backbone, such as VDC / IE. In some instances, one or more doses of the selected chemotherapy backbone may be lowered compared to the typical standard-of-care dose. As shown in Example 4, a combination induction chemotherapy comprising a standard-of-care chemotherapeutic backbone and an anti-GD2 antibody can yield an overall better response compared with either the standard-of-care chemotherapeutic backbone or anti-GD2 antibody when used separately. Consequently, the invention may be used to reduce chemotherapeutic burden. In some embodiments, VDC is administered in the first cycle of combination induction chemotherapy and IE is administered in the second cycle of combination induction chemotherapy. In such an embodiment, the cycles alternate between VDC and IE treatment until the completion of the combination induction chemotherapy. The anti- GD2 antibody may be administered simultaneously or sequentially with VDC or IE. The antibody may be administered continuously over several days, for example, 3, 4, 5, 6, or 7 days.

[0149] Anti-GD2 Antibodies

[0150] By "antibody" we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bi-specific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen binding fragments and derivatives of the same. The term also includes antibody-like molecules which may be produced using phage-display techniques or other random selection techniques for molecules. The term also includes all classes of antibodies, including IgG, IgA, IgM, IgD, and IgE. Also included for use in the invention are antibody fragments such as Fab, F(ab')2, Fv, Fab', scFv (single-chain variable fragment), or di- scFv and other fragments thereof that retain the antigen-binding site. Similarly, the term "antibody" includes genetically engineered derivatives of antibodies such as single-chain Fv molecules (scFv) and single-domain antibodies (dAbs).

[0151] It will be appreciated that a suitable anti-GD2 antibody may encompass a bi-specific or multi-specific antibody, wherein one or more antibody-binding moieties or CDR sequences (for example, those described in Table 4) would be capable of binding to the GD2 antigen. Other suitable sequences for anti-GD2 antibodies can include those described in Moghimi et al ( / Vat Commun 12, 511 (2021). It will be further appreciated that the use of other biologies comprising bi-specific or multi-specific properties may also be suitable for carrying out the invention, for example, bi-specific or multi-specific CAR-T cell therapies, which bind to GD2 and at least one other antigen presented on Ewing sarcoma cells or on a cell type with an immunological role.

[0152] It will be further appreciated that a suitable anti-GD2 antibody may encompass antibody-drug conjugates (ADCs). Suitable anti-GD2 antibody conjugates are described in WO 2023 / 172968, for example, "Molecule 1", also referred to as M3554, which comprises an anti-GD2 antibody conjugated to a 0-glucuronide linker and Exatecan growth inhibitory agent. For the avoidance of doubt, a drug conjugated to the antibody as an ADC does not constitute a chemotherapeutic agent in the context of the invention.

[0153] In addition, it will be understood by the skilled person that the treatment described herein may further comprise a cell-based therapy, for example, an allogeneic natural killer (NK) cell based therapy.

[0154] Preferred antibodies are chimeric, such as mouse-human chimeric antibodies, CDR- grafted antibodies, humanised antibodies, or human antibodies. For example, the anti- GD2 antibody may be a chimeric, humanized or CDR grafted anti-GD2 antibody. In an embodiment, the anti-GD2 antibody is a chimeric anti-GD2 antibody. Unlike rodent antibodies, chimeric antibodies are expected to be less immunogenic and would therefore be preferable in carrying out the methods of the invention. Although the antibody may be a polyclonal antibody, it is preferred if it is a monoclonal antibody, or that the antigen-binding fragment is derived from a monoclonal antibody. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies; A manual of techniques" (Zola, H. CRC Press. 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Application" (Hurrell, SGR. CRC Press). The antibodies may be human antibodies in the sense that they have the amino acid sequence of human antibodies with specificity for GD2; however, it will be appreciated that they may be prepared using methods known in the art that do not require immunisation of humans. Suitable antibodies may be prepared from transgenic mice which contain human immunoglobulin loci, as described in "Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery" (Lee, H et al. Nat Biotechnol. 2014. 32, 356-363).

[0155] Suitably prepared non-human antibodies can be "humanised" in known ways, for example, by inserting the CDR regions of mouse antibodies into the framework of human antibodies. Chimeric antibodies are discussed in Neuberger et al (1998, 8th International Biotechnology Symposium Part 2, 792-799).

[0156] It will be appreciated by persons skilled in the art that the binding specificity of an antibody or antigen-binding fragment thereof is conferred by the presence of complementarity determining regions (CDRs) within the variable regions of the constituent heavy and light chains. As discussed below, in a particularly preferred embodiment of the antibodies and antigen-binding fragments, binding specificity for GD2 is conferred by the presence of one or more and typically all six of the CDR amino acid sequences defined herein. Each CDR.3 of the heavy and light chain variable regions is particularly important in determining binding specificity.

[0157] Suitably, the antibody or antigen-binding fragment comprises an antibody Fc region. It will be appreciated by the skilled person that the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD, or IgE). For example, the Fc region may be from an IgGl, IgG2, IgG3, or IgG4 antibody. Advantageously, however, the Fc region is from an IgGl antibody. It is preferred that the antibody or antigen-binding fragment is an IgG molecule, or is an antigen-binding fragment or variant of an IgG molecule. The Fc region may include a mutation to reduce complement binding, such as the K322A point mutation or other antibody sequences as described in WO 2023 / 172968 Al.

[0158] Anti-GD2 antibodies which may be used in the invention include, but are not limited to, dinutuximab, dinutuximab beta, and naxitamab. Fragments of any of these antibodies may also be used. Suitably, the anti-GD2 antibody or fragment is a chimeric, humanized or CDR grafted antibody or fragment thereof.

[0159] Naxitamab is FDA-approved for treatment of high-risk neuroblastoma in combination with GM-CSF for pediatric patients one year of age and older and adult patients with relapsed or refractory high-risk neuroblastoma in the bone or bone marrow demonstrating a partial response, minor response, or stable disease to prior therapy. Efficacy was evaluated in patients with relapsed or refractory neuroblastoma in the bone or bone marrow enrolled in two single-arm, open-label trials: Study 201 (NOT 03363373) and Study 12-230 (NOT 01757626).

[0160] Dinutuximab and dinutuximab beta are approved monoclonal antibodies for postconsolidation therapy in paediatric patients with high-risk neuroblastoma. Dinutuximab is approved by the FDA and sold under the name Unituxin®. It is administered to high- risk neuroblastoma patients who achieve at least a partial response to prior first-line multiagent, multimodality therapy. In that context, administration is typically performed intravenously over 10 or 20 hours for 4 consecutive days for up to 5 cycles. Efficacy in that context was evaluated in a paediatric study of 226 patients in a two single-arm trial. In that context, the treatment is typically given in conjunction with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 and isotretinoin. Dinutuximab beta is approved in Europe under the name Qarziba® and approval is pending in the US and other countries. It is also used as a second line treatment for children with high-risk neuroblastoma; in that context it was tested and is used with a longer and slower dosing regime, and is given on its own, although it may be combined with IL-2 if a stronger immune response is needed.

[0161] In an embodiment, the anti-GD2 antibody is Dinutuximab or Dinutuximab beta. In an embodiment, the anti-GD2 antibody is Dinutuximab beta.

[0162] Nucleotide sequences encoding dinutuximab and dinutuximab beta are provided below as SEQ ID NO: 1 and SEQ ID NO: 2. The skilled person will appreciate that these sequences can be inserted into any suitable expression vector for production of the antibody.

[0163] In an embodiment, the antibody has the heavy chain sequence SEQ ID NO: 19 and the light chain sequence SEQ ID NO: 18.

[0164] SEQ ID NO: 1 (Light chain DNA sequence):

[0165] ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACTGGAGA AATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGATCTAGTCAGAGTCTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGC AGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGGTC CCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCG GTGCTGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCG CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0166] SEQ ID NO: 2 (Heavy chain DNA sequence):

[0167] ATGGGATGGACCTGGATCTTTATTTTAATCCTGTCGGTAACTACAGGTGTCCACTCTGAGGTC CAACTGCTGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGATGATATCCTGCAA GGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGA GCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTGGAACTAGCTACAACCAGAAGTTC AAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGA GCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAA GGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA

[0168] Protein sequences of the light (SEQ ID NO: 3) and heavy (SEQ ID NO:4) chains of dinutuximab or dinutuximab beta including signal peptide sequence are shown below. The signal peptide sequence (shown in bold underlined font) is removed during post translational processing and does not form part of the final recombinant protein, which therefore has a light chain sequence of SEQ ID NO: 18 and a heavy chain sequence of SEQ ID NO: 19. In any event, the skilled person will appreciate how to determine the identity of the signal peptide sequence and the positioning of the cleavage site. For example, the cleavage site for the light chain sequence (SEQ ID NO: 3) may be between positions 20 and 21 (Gly20-Glu21); the cleavage site for the heavy chain sequence (SEQ ID NO:4) may be between positions 19 and 20 (Serl9-Glu20). Alternative signal peptide sequences may also be used.

[0169] SEQ ID NO:3 (Light chain protein sequence with signal peptide):

[0170] MEAPAOLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYL QKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGA GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:4 (Heavy chain protein sequence with signal peptide):

[0171] MGWTWIFILILSVTTGVHSEVOLLQSGPELEKPGASVMISCKASGSSFTGYN M N WVRQN IG KSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQ GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

[0172] SEQ ID NO:18 (Final light chain protein sequence):

[0173] EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0174] SEQ ID NO:19 (Final heavy chain protein sequence):

[0175] EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQK FKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0176] Table 4 shows some exemplary CDR sequences of antibodies that may be suitable for the invention. The sequences are given according to three different definitions that the skilled person is aware how to use. The Kabat definition is based on sequence variability. The Chothia definition is based on the location of the structural loop regions. The IMGT numbering scheme was originally based on alignment of germ-line V genes, spanning from FR1 to the beginning of the CDR3, and was later extended to cover the entire variable region; the numbering runs from 1 to 128 based on the V- gene sequence alignment, with an insertion point only between positions 111 and 112 in the CDR3 for lengths exceeding 13 amino acids. Table 4: CDR sequences for variable heavy (VH) and variable light (VL) chain regions of an antibody (dinutuximab or dinutuximab beta) according to three different definitions (IMGT, Kabat, Chothia) Suitable antibodies and fragments are also described in WO 2013 / 189554 Al.

[0177] Suitably, the antibody or fragment is a chimeric, humanized or CDR grafted antibody or fragment thereof comprising a light chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; and a heavy chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 8, SEQ ID NO:, 9 and SEQ ID NO: 10, respectively, and a constant region derived from a human IgG antibody or a humanised IgG antibody. Suitably, the antibody or fragment is a chimeric, humanized or CDR grafted antibody or fragment thereof comprising a light chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 7, respectively; and a heavy chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 13, SEQ ID NO:, 14 and SEQ ID NO: 15, respectively, and a constant region derived from a human IgG antibody or a humanised IgG antibody.

[0178] Suitably, the antibody or fragment is a chimeric, humanized or CDR grafted antibody or fragment thereof comprising a light chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 7, respectively; and a heavy chain variable region in which CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 15, respectively, and a constant region derived from a human IgG antibody or a humanised IgG antibody.

[0179] The protein component of Dinutuximab beta consists of 2 light chains (220 amino acids) and 2 heavy chains (443 amino acids) and is of the IgGl subclass. The monoclonal antibody incorporates human constant regions for the heavy chain IgGl and the kappa light chain, along with the mouse variable regions targeted specifically against human GD2. The relative molecular mass of the intact antibody is approximately 150,000 daltons. The encoding nucleotide sequences and the amino acid sequences of chimeric anti-GD2 antibody dinutuximab beta are also provided in WO 2013 / 189554 Al (SEQ ID NOs: l-4 therein). Methods of manufacture and formulation of dinutuximab beta for clinical use are described in the European Public Assessment Report (EPAR) of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) for Dinutuximab beta Apeiron (EMA / 263814 / 2017, 23 March 2017).

[0180] A preparation comprising an anti-GD2 antibody, such as a dinutuximab beta, may further comprise salts and water for injection (WFI). In one embodiment, the preparation comprising an anti-GD2 antibody, such as dinutuximab beta, may further comprise a buffer, e.g., phosphate-buffered saline, comprising said salts and WFI. A preparation comprising an anti-GD2 antibody, such as dinutuximab beta, may further comprise stabilizing agents, preservatives and other carriers or excipients. The preparation comprising an anti-GD2 antibody, such as a dinutuximab beta, may be freeze-dried and reconstituted for use. In one embodiment, the preparation comprising an anti-GD2 antibody, such as dinutuximab beta, does not comprise preservatives and other excipients. The preparation comprising an anti-GD2 antibody, such as dinutuximab beta, may be added to an infusion bag, e.g., an infusion bag containing 100 mL NaCI 0.9% and 5 mL human serum albumin 20%.

[0181] Typical properties of dinutuximab beta are further shown in Table 5.

[0182] Table 5: Properties of dinutuximab beta

[0183] In an embodiment of any of the aspects of the invention, the administration to the patient of the anti-GD2 antibody or fragment is by intravenous administration, optionally by infusion, optionally by an infusion pump.

[0184] Suitably, the anti-GD2 antibody is administered by using a mini-pump. Suitably, the mini-pump is a commercially available mini-pump.

[0185] In an embodiment, the anti-GD2 antibody is administered as a continuous infusion over 24 hours and over consecutive days until the dose for the cycle has been administered in full. An antibody fragment is to be administered at an equivalent fragment dose having an equivalent antagonistic effect on GD2 to the whole antibody from which the fragment is derived. The equivalent fragment dose may be calculated according to the fragment molecular weight compared to the molecular weight of the whole antibody, also referred to as parent antibody. For example, if a given antibody has a molecular weight of 150 kDa, and a Fab fragment has a molecular weight of 50 kDa, then a fragment dose that is one third of the antibody dose should provide an equivalent antagonistic effect on GD2. The equivalent antagonistic effect on GD2 may also be determined according to the amount of GD2 that the fragment can specifically bind to, compared to the amount of GD2 that the parent antibody can specifically bind to. These amounts may be determined by various assays, including ELISA.

[0186] In an embodiment, the anti-GD2 antibody, especially dinutuximab or dinuxutimab beta, is administered at a dose of from about 1 mg / m2 / d to about 20 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d to about 10 mg / m2 / d, such as at a dose from about 5 mg / m2 / d to about 10 mg / m2 / d (such as from about 7.5 mg / m2 / d to about 10 mg / m2 / d) or at a dose from about 4 mg / m2 / d to about 8 mg / m2 / d (such as from about 5 mg / m2 / d to about 7.5 mg / m2 / d). Suitably, the anti-GD2 antibody is administered at a dose of about 1 mg / m2 / d, 2 mg / m2 / d, 3 mg / m2 / d, 4 mg / m2 / d, 5 mg / m2 / d, 6 mg / m2 / d, 7 mg / m2 / d, 8 mg / m2 / d, 9 mg / m2 / d, 10 mg / m2 / d, 11 mg / m2 / d, 12 mg / m2 / d, 13 mg / m2 / d, 14 mg / m2 / d, 15 mg / m2 / d, 16 mg / m2 / d, 17 mg / m2 / d, 18 mg / m2 / d, 19 mg / m2 / d, or 20 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d, 5 mg / m2 / d, 6 mg / m2 / d, 7 mg / m2 / d, 8 mg / m2 / d, or 9 mg / m2 / d, to about 10 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d, or 5 mg / m2 / d, to about 6 mg / m2 / d, 7 mg / m2 / d, 8 mg / m2 / d, 9 mg / m2 / d, or 10 mg / m2 / d.

[0187] The dose may be adjusted at one or more points during the treatment, or across different treatment doses, or across different treatment or therapy cycles. In an embodiment, the dose of anti-GD2 antibody, especially dinutuximab or dinutuximab beta, is selected from 1 mg / m2 / d to about 20 mg / m2 / d (such as from about 5 mg / m2 / d to about 10 mg / m2 / d, such as 5 mg / m2 / d, 7.5 mg / m2 / d, 10 mg / m2 / d) according to the subject's tolerance.

[0188] The use of immunotherapy agents, including antibodies, can trigger cytokine release syndrome (CRS). Cytokine release syndrome, and Cytokine storms are life-threatening systemic inflammatory syndromes involving elevated levels of circulating cytokines and immune-cell hyperactivation that can be triggered by various therapies, pathogens, cancers, autoimmune conditions, and monogenic disorders. Accordingly, the dose of anti-GD2 antibody may be adjusted throughout treatment or therapy cycles.

[0189] In an embodiment, the anti-GD2 antibody, especially dinutuximab or dinutuximab beta, especially dinutuximab beta, is administered at a dose of about 10 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 9 mg / m2 / d to about 11 mg / m2 / d, such as from about 9.5 mg / m2 / d to about 10.5 mg / m2 / d.

[0190] In an embodiment, the anti-GD2 antibody, especially dinutuximab or dinutuximab beta, especially dinutuximab beta, is administered at a dose of about 7.5 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 6 mg / m2 / d to about 9 mg / m2 / d, such as from about 7 mg / m2 / d to about 8 mg / m2 / d.

[0191] In an embodiment, the anti-GD2 antibody, especially dinutuximab or dinutuximab beta, especially dinutuximab beta is administered at a dose of about 5 mg / m2 / d. Suitably, the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d to about 7 mg / m2 / d, or from about 4 mg / m2 / d to about 6 mg / m2 / d, such as from about 4.5 mg / m2 / d to about 5.5 mg / m2 / d.

[0192] Suitably, the anti-GD2 antibody, especially dinutuximab or dinutuximab beta, especially dinutuximab beta, is administered at a dose of 5 mg / m2 / d, 7.5 mg / m2 / d, or 10 mg / m2 / d.

[0193] The anti-GD2 antibody may be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days per treatment cycle. For example, the anti-GD2 antibody may be administered for 4 days per treatment cycle, such as in the typical treatment with dinutuximab (Unituxin®). Alternatively, the anti-GD2 antibody may be administered for 5 days per treatment cycle, such as in the typical treatment with dinutuximab beta (Qarziba®), particularly when administered non-continuously. Alternatively, the anti-GD2 antibody may be administered for 10 days per treatment cycle, such as in the typical treatment with dinutuximab beta (Qarziba®), particularly when administered by continuous infusion.

[0194] The anti-GD2 antibody may be administered by continuous IV infusion at a dose of 10 mg / m2 / day for up to 12 days per treatment cycle. Suitably, the antibody may be administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days per treatment cycle. For example, the anti-GD2 antibody may be administered for 5 days per treatment cycle. Alternatively, the anti-GD2 antibody may be administered for 10 days per treatment cycle. In some embodiments, the anti-GD2 antibody is administered at a dose of 10 mg / m2 / day for 10 days per treatment cycle.

[0195] In one particular embodiment, dinutuximab beta is administered at a dose of 10 mg m2 / day for 7 days per treatment cycle resulting in a total of dose 70 mg / m2per cycle.

[0196] In another particular embodiment, dinutuximab beta is administered at a dose of 10 mg m2 / day for 10 days per treatment cycle resulting in a total dose of 100 mg / m2per cycle.

[0197] In one particular embodiment, dinutuximab beta is administered at a dose of 10 mg / m2 / day for 10 days per treatment cycle, particularly by continuous IV infusion. For example, such administration may be for 6-12 cycles, such as 6 cycles or 12 cycles.

[0198] It will be appreciated that the total dose of dinutuximab beta per cycle may change in each treatment cycle to accommodate the subject's response and / or in response to clinically relevant factors.

[0199] By "cumulative dose", we mean the total dose that is administered (i) during a cycle, or (ii) during a phase or (iii) during the overall treatment time. The skilled person will understand that the cumulative dose is equal or larger for (iii) than for (ii), and equal or larger for (ii) than for (i). Preferably, "cumulative dose" is defined by (iii). The units of dose are expressed in mg / m2, where the area (in m2) refers to the patient's body surface area (BSA). For example, if a patient has a body surface area of 0.7 m2, then a cumulative dose of 3000 mg / m2would be 2100 mg.

[0200] In some embodiments, the anti-GD2 antibody (such as dinutuximab or dinutuximab beta) may be administered at a cumulative dose of up to 3000 mg / m2, such as at a cumulative dose of at least 100 mg / m2, such as from 100 to 200 mg / m2, from 200 to

[0201] 300 mg / m2, from 300 to 400 mg / m2, from 400 to 500 mg / m2, from 500 to 600 mg / m2, from 600 to 700 mg / m2, from 700 to 800 mg / m2, from 800 to 900 mg / m2, from 900 to 1000 mg / m2, from 1000 to 1100 mg / m2, from 1100 to 1200 mg / m2, from 1200 to 1300 mg / m2, from 1300 to 1400 mg / m2, from 1400 to 1500 mg / m2, from 1500 to

[0202] 1600 mg / m2, from 1600 to 1700 mg / m2, from 1700 to 1800 mg / m2, from 1800 to

[0203] 1900 mg / m2, from 1900 to 2000 mg / m2, from 2000 to 2100 mg / m2, from 2100 to

[0204] 2200 mg / m2, from 2200 to 2300 mg / m2, from 2300 to 2400 mg / m2, from 2400 to

[0205] 2500 mg / m2, from 2500 to 2600 mg / m2, from 2600 to 2700 mg / m2, from 2700 to 2800 mg / m2, from 2800 to 2900 mg / m2, or from 2900 to 3000 mg / m2. Optionally, the cumulative dose is up to 850 mg / m2, up to 950 mg / m2, up to 1050 mg / m2, up to 1250 mg / m2, up to 1350 mg / m2, up to 1450 mg / m2, up to 1550 mg / m2, or up to 2050 mg / m2.

[0206] The anti-GD2 antibody may have a predetermined overall patient dose and the anti- GD2 antibody may be administered in cycles until the predetermined overall patient dose has been administered. By "predetermined overall patient dose" we mean that a dose of anti-GD2 antibody may be determined before or during the treatment period. For example, the predetermined dose may be a certain cumulative dose (defined by (iii) above) which is determined before the first treatment dose of the antibody is given. Alternatively, the predetermined dose may be a cumulative dose determined after the first treatment dose of the antibody. The predetermined dose may apply per cycle, or for the whole treatment period (i.e., across multiple cycles).

[0207] In an additional embodiment according to the first aspect of the invention, the antibody may be further administered in one or more antibody treatment cycles without chemotherapy. For example, after completion of the combined induction chemotherapy, the antibody may be administered in one or more further cycles of antibody treatment. In one particular embodiment, after completion of the combined induction chemotherapy, the antibody may be administered in 6 further cycles of antibody treatment. In a non-limiting example, 6 cycles of combination induction chemotherapy may be administered to a subject followed by a further 6 cycles of antibody treatment. In another non-limiting example, 4 cycles of combination induction chemotherapy may be administered to a subject followed by a further 10-12 cycles of antibody treatment.

[0208] It will be appreciated that some of the combined induction chemotherapy regimens described in this aspect will be particularly effective at treating relapsed or high-risk metastatic GD2-positive Ewing sarcoma. This may include specific combinations of an anti-GD2 antibody and one or more chemotherapeutic agents. It will also be understood that the treatments described herein will be applicable to the treatment of subjects experiencing a second or subsequent relapse of GD2-positive Ewing Sarcoma.

[0209] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of TEMIRI (Temozolomide & Irinotecan) or TOPOCYLO (Topotecan & Cyclophosphamide) chemotherapy. In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of TEMIRI (Temozolomide & Irinotecan) or TOPOCYLO (Topotecan & Cyclophosphamide) chemotherapy, wherein the treatment comprises at least 6 21-day cycles, for example, 6, 7, 8, 9, 10, 11 or 12 cycles.

[0210] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of TEMIRI (Temozolomide & Irinotecan) or TOPOCYLO (Topotecan & Cyclophosphamide) chemotherapy, wherein the treatment comprises at least 6 21-day cycles; and wherein the anti-GD2 antibody is dinutuximab beta.

[0211] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of TEMIRI (Temozolomide & Irinotecan) or TOPOCYLO (Topotecan & Cyclophosphamide) chemotherapy, wherein the treatment comprises at least 6 21-day cycles; and wherein the anti-GD2 antibody is dinutuximab beta; and wherein the anti-GD2 antibody is administered by continuous infusion at a dose of 10 mg / m2 / d for 10 days per cycle.

[0212] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of TEMIRI (Temozolomide & Irinotecan) or TOPOCYLO (Topotecan & Cyclophosphamide) chemotherapy, wherein the combination chemotherapy comprises at least 6 21-day cycles followed by at least 6 further cycles of the anti-GD2 antibody therapy.

[0213] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of HD IFOS (high dose ifosfamide).

[0214] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of HD IFOS (high dose ifosfamide) chemotherapy, wherein the treatment comprises at least 4 21-day cycles, for example, 4, 5, 6, 7, 8, 9 or 10 cycles.

[0215] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of HD IFOS (high dose ifosfamide) chemotherapy, wherein the treatment comprises at least 4 21-day cycles; and wherein the anti-GD2 antibody is dinutuximab beta.

[0216] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of HD IFOS (high dose ifosfamide) chemotherapy, wherein the treatment comprises at least 4 21-day cycles; and wherein the anti-GD2 antibody is dinutuximab beta; and wherein the anti-GD2 antibody is administered by continuous infusion at a dose of 10 mg / m2 / d for 10 days per cycle.

[0217] In one embodiment, the treatment of GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) the administration of HD IFOS (high dose ifosfamide) chemotherapy, wherein the combination chemotherapy comprises at least 4 21-day cycles followed by at least 10 further cycles of the anti-GD2 antibody therapy, such as 10-12 further cycles of the anti-GD2 antibody therapy.

[0218] In the above embodiments relating to TEMIRI, TOPOCYCLO or HD IFOS, the skilled person will understand that the number of days per cycle may be varied, for example to replace the 21-day cycles with 28-day cycles.

[0219] Combination Consolidation Chemotherapy

[0220] In the second aspect of the invention, there is provided a method of treating GD2- positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy according to the first aspect of the invention, followed by a combination consolidation chemotherapy of:

[0221] (i) administration to the subject of an anti-GD2 antibody; and

[0222] (ii) administration to the subject of at least one treatment dose of consolidation chemotherapy with one or more chemotherapeutic agent(s). Optionally, the schedule of administration of the antibody in the combination induction chemotherapy may be continued in the same manner in the combination consolidation chemotherapy so that there is no disruption or discontinuity of the antibody doses between the induction and consolidation therapies.

[0223] Typically, the antibody administered in the combined consolidation chemotherapy may be the same as the antibody administered in the combined induction chemotherapy. Alternatively, the antibody administered in the combined consolidation chemotherapy may be different to the antibody administered in the combined induction chemotherapy.

[0224] The one or more chemotherapeutic agent(s) in the consolidation chemotherapy and associated embodiments may be as described in the section above entitled "Chemotherapeutic agents", mutatis mutandis.

[0225] Consolidation therapy may comprise one or more chemotherapeutic agents selected from one or more of vincristine (V), actinomycin D (A), cyclophosphamide (C or CYCLO), ifosfamide (I), temozolomide (TEM), irinotecan (IRI), and / or topotecan (TOPO). For instance, VAI and VAC may be administered in alternate cycles in accordance with the Euro Ewings trial described herein.

[0226] Cycles of VAI may be given at 21 day intervals (+ / - 3 days) and on haematological recovery to ANC >1.0xl09 / L, platelets >80xl09 / L. VAI may be administered in accordance with Table 6.

[0227] Table 6: VAI consolidation chemotherapy Patients whose surface area is > 2 m2may have their doses capped and calculated with a SA of 2 m2. Granulocyte-colony stimulating factor = G-CSF.

[0228] Cycles of VAC may be given at 21 day intervals (+ / - 3 days) and on haematological recovery to ANC >1.0xl09 / L, platelets >80xl09 / L.

[0229] VAC may be administered in accordance with Table 7.

[0230] Table 7: VAC consolidation chemotherapy

[0231] Patients whose SA is > 2 m2should have their doses capped and calculated with a SA of 2 m2.

[0232] In one embodiment, the antibody in the combination consolidation chemotherapy is administered in a first treatment dose:

[0233] (a) simultaneously with one or more of the chemotherapeutic agent(s) in the first cycle of the consolidation chemotherapy;

[0234] (b) as sequential administration with the one or more chemotherapeutic agent(s) in the first cycle of the consolidation chemotherapy;

[0235] (c) simultaneously with one or more of the chemotherapeutic agent(s) in a second or subsequent cycle of the consolidation chemotherapy, such as in a second, third, fourth, fifth, sixth, or subsequent cycle; or

[0236] (d) as sequential administration with the one or more chemotherapeutic agent(s) in a second or subsequent cycle of the consolidation chemotherapy, such as in the second, third, fourth, fifth, sixth, or subsequent cycle. References to simultaneous and sequential administration are to be interpreted according to the definitions established in the first aspect of the invention.

[0237] In an embodiment of the second aspect of the invention, administered simultaneously means beginning administering the antibody within the dosing period of the consolidation chemotherapy. For instance, if the one or more chemotherapeutic agent(s) are administered over 5 days, the administration of the antibody may begin on the first, second, third, fourth or fifth day in that dosing period.

[0238] In particular, administered simultaneously may include beginning administering the antibody during the dosing period of the one or more of the chemotherapeutic agent(s) of the consolidation chemotherapy, on the same calendar day.

[0239] In an embodiment of the second aspect of the invention, administered simultaneously means administering the antibody within the dosing period of the one or more chemotherapeutic agent(s) of the consolidation chemotherapy contemporaneously to the extent that the antibody begins to be applied within one hour of a dose of one or more of the chemotherapeutic agent(s).

[0240] In one embodiment, the antibody in the combination consolidation chemotherapy is administered:

[0241] (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy;

[0242] (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy;

[0243] (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy; or

[0244] (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy.

[0245] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy. This means that with each dosing period of the one or more chemotherapeutic agent(s), a treatment dose of antibody is administered simultaneously. The context by which the antibody is administered simultaneously may vary independently in each cycle. For example, in one cycle of consolidation chemotherapy, the antibody may be administered on the same calendar day as one or more of the chemotherapeutic agent(s). In another cycle of the consolidation chemotherapy, the antibody may be administered within one hour of administering one or more chemotherapeutic agent(s).

[0246] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy. This means that following each dosing period of the one or more chemotherapeutic agent(s), a treatment dose of antibody is administered sequentially. The context by which the antibody is administered sequentially can vary independently in each cycle. For example, in one cycle of consolidation chemotherapy, the antibody may be administered one calendar day after the administration of the chemotherapeutic agent(s). In another cycle of the induction chemotherapy, the antibody may be administered two or more calendar days after the administration of the chemotherapeutic agent(s).

[0247] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy. As a non-limiting example, if the first dose of antibody is administered in the second treatment cycle of the combination consolidation chemotherapy which comprises 6 treatment cycles, the antibody would be administered simultaneously in the third, fourth, fifth, and sixth treatment cycles.

[0248] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy. As a non-limiting example, if the first dose of antibody is administered in the second treatment cycle of the combination consolidation chemotherapy which comprises 6 treatment cycles, the antibody would be administered sequentially in the third, fourth, fifth, and sixth treatment cycles.

[0249] Alternatively in another embodiment, the administration of the antibody in the combined consolidation chemotherapy may be discontinued after one or more treatment cycles, such as after two, three, four, five or more treatment cycles. This can be combined with any embodiment relating to simultaneous or sequential administration of the antibody.

[0250] Without wishing to be bound by theory, when the antibody in the combination consolidation chemotherapy is administered in a first treatment dose simultaneously with a first treatment dose of one or more chemotherapeutic agent(s) of the consolidation chemotherapy, there may be a faster response to treatment. Alternatively and without wishing to be bound by theory, when the first dose of the antibody is given later in treatment, there may be reduced side effects, such as a reduced risk of the subject developing a potential TLS or developing CRS. In some instances, the dosing strategy adopted in the induction chemotherapy will be used in the subsequent consolidation chemotherapy. Accordingly, the anti-GD2 antibody in the combination consolidation chemotherapy is administered in a first treatment dose simultaneously with a first treatment dose of one or more chemotherapeutic agent(s) of the consolidation chemotherapy or the first dose of the antibody is given later in treatment. In other instances, diagnostic indicators or clinical practice may suggest a different antibody dosing strategy to reduce the risk of the subject developing a potential TLS or developing CRS. The skilled person would recognise the clinical indicators of TLS and / or CRS and choose an appropriate option, which is in line with the recommended standard of care.

[0251] Suitably, the chemotherapeutic agents of the consolidation chemotherapy described herein may be selected from one or more of the following: vincristine, ifosfamide, cyclophosphamide, doxorubicin, etoposide, temozolomide, irinotecan, and topotecan. In some instances, actinomycin D may be included in the consolidation chemotherapy. The chemotherapeutic agents and schedule thereof in the consolidation chemotherapy may be the same or different to those in the induction chemotherapy. The doses of individual chemotherapeutic agents described herein for consolidation therapy or induction chemotherapy can also be used vice versa.

[0252] In an embodiment, the chemotherapeutic agents administered to the patient are in combinations selected from:

[0253] (i) vincristine, doxorubicin, and cyclophosphamide (VDC);

[0254] (ii) ifosfamide, and etoposide (IE);

[0255] (iii) vincristine, ifosfamide, doxorubicin, and etoposide (VIDE);

[0256] (iv) vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide (VDCIE);

[0257] (v) vincristine, actinomycin D and ifosfamide (VAI);

[0258] (vi) temozolomide and irinotecan (TEMIRI); and / or (vii) topotecan and cyclophosphamide (TOPOCYCLO).

[0259] In one embodiment, vincristine may be administered in the consolidation chemotherapy at a dose of at least 1.5 mg / m2. Vincristine may be administered in an IV push orthrough short infusion. The maximum single dose of vincristine administered per cycle may be 2 mg. Suitably, a single dose of vincristine used per cycle can include 2, 1.9, 1.9, 1.7, 1.6, or 1.5 mg.

[0260] In one embodiment, actinomycin D may be administered in the consolidation chemotherapy at a dose of at least 0.75 mg / m2 / d. Actinomycin D may be administered through an IV push. The maximum single dose of actinomycin D administered per cycle may be 1.5 mg. Suitably, a single dose of actinomycin D used per cycle can include 1.5, 1.25, 1, or 0.7 mg.

[0261] In one embodiment, ifosfamide may be administered in the consolidation chemotherapy at a dose of at least 3 g / m2 / d. Ifosfamide may be administered in an IV infusion over 1-3 hours.

[0262] In one embodiment, cyclophosphamide may be administered in the consolidation chemotherapy at a dose of at least 1500 mg / m2. Cyclophosphamide may be administered in an IV infusion over 1-3 hours.

[0263] In one embodiment, actinomycin D may be administered in the consolidation chemotherapy at a dose of at least 0.75 mg / m2 / d. Actinomycin D may be administered through an IV push. The maximum single dose of actinomycin D administered per cycle may be 1.5 mg. Suitably, a single dose of actinomycin D used per cycle can include 1.5, 1.25, 1, or 0.7 mg.

[0264] In one embodiment, temozolomide may be administered in the consolidation chemotherapy at a dose of at least 75 mg / m2. Temozolomide may be administered by intravenous infusion or oral administration. Temozolomide may be provided as a 100 mg lyophilized powder in single-dose vial for reconstitution or in capsules containing 5 mg, 20 mg, 100 mg, 140 mg, 180 mg, or 250 mg of the agent.

[0265] In one embodiment, topotecan may be administered at a dose of at least 1.5 mg / m2. Topotecan may be administered by IV infusion, such as for 30 minutes daily for up to 5 days. Topotecan may be provided as a single-use vial containing 4 mg of topotecan free base as a 4 mg / 4 mL (1 mg / ml final concentration). In one embodiment, irinotecan may be administered at a dose of at least 125 mg / m2. Irinotecan may be administered by intravenous infusion, such as for 90 minutes daily. Irinotecan may be provided as single use vials, containing, 40, 100, or 300 mg of an irinotecan hydrochloride solution.

[0266] Optionally, Granulocyte colony stimulating factor (G-CSF) may be administered as a supplement during consolidation chemotherapy. G-CSF is a type of growth factor that stimulates bone marrow to produce more white blood cells and may be included in a consolidation therapy to reduce the risk of infection.

[0267] Optionally, zoledronic acid, also known as a bisphosphonate, may be administered as a chemotherapeutic agent in consolidation chemotherapy. In one embodiment, zoledronic acid may be administered in the consolidation chemotherapy at a dose of at least 0.05 mg / kg by IV infusion. In some instances, the dose of zoledronic acid may be reduced or fully stopped in accordance with nephrotoxicity guidelines. A suitable clinical indicator may be reduced calculated creatinine clearance GFR (ml / min / 1.73 m2).

[0268] In an embodiment, two or more chemotherapeutic agent combinations are administered in alternating cycles of consolidation chemotherapy. In an embodiment, IE and VC are administered in alternating cycles of consolidation chemotherapy. In an embodiment, VDC and IE are administered in alternating cycles of consolidation chemotherapy.

[0269] In a further embodiment, at least two cycles of consolidation chemotherapy are administered; optionally, wherein at least 3 cycles of consolidation chemotherapy are administered; further optionally, wherein at least 5 cycles of consolidation chemotherapy are administered. For example, the consolidation chemotherapy may comprise 2, 3, 4, or 5 cycles.

[0270] Depending on the outcome of the combined induction chemotherapy, the dose of anti- GD2 antibody administered in the combination consolidation chemotherapy may be lowered. For example, the dose may be lowered by up to 50% compared to one or more dose(s) administered during combined induction chemotherapy. For example, the anti-GD2 antibody dose, in each independent treatment cycle or across multiple cycles, may be lowered by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% compared to the dose or a particular dose administered in the combined induction chemotherapy. Accordingly, the dose administered to the patient may be 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50% compared to the dose or a particular dose administered in the combined induction chemotherapy.

[0271] In one embodiment, the antibody is administered in the combination consolidation chemotherapy at a dose that is:

[0272] (i) lower than the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy; or

[0273] (ii) at a dose that is 75% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy;

[0274] (iii) at a dose that is 50% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy; or

[0275] (iv) the same as the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy.

[0276] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (i) at a lower dose than at which the anti-GD2 antibody is administered in the combination induction chemotherapy. For example, the dose of anti-GD2 antibody administered in the combination consolidation chemotherapy, in a single dose or across multiple treatment cycles, may be 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 mg / m2 / d. The dose for each treatment, cycle, or individual dose may be independently lowered compared to the dose administered in combination induction chemotherapy.

[0277] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (ii) at a dose that is 75% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy. This may refer to a single dose of anti-GD2 antibody administered in the combination induction chemotherapy or an average of doses across multiple cycles. In some instances, the dose is lowered to about 75% of the dose at which anti-GD2 antibody is administered in the combination induction chemotherapy.

[0278] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (iii) at a dose that is 50% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy. This may refer to a single dose of anti-GD2 antibody administered in the combination induction chemotherapy or an average of doses across multiple cycles. In some instances, the dose is lowered to about 50% of the dose at which anti-GD2 antibody is administered in the combination induction chemotherapy.

[0279] In an embodiment, the antibody in the combination consolidation chemotherapy is administered (iv) at the same dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy. This may refer to a single dose of anti-GD2 antibody administered in the combination induction chemotherapy or an average of doses across multiple cycles.

[0280] Alternatively, depending on the outcome of a combination induction chemotherapy, the dose of the antibody administered in the combination consolidation chemotherapy may be increased. For example, the dose may be increased by up to or more than 100% compared to one or more dose(s) administered during combination induction chemotherapy. For instance, the dose of the antibody may be increased by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150% compared to one or more dose(s) of the antibody administered during the combination induction chemotherapy.

[0281] In an additional embodiment according to the second aspect of the invention, the antibody may be further administered in one or more antibody treatment cycles without chemotherapy. For example, after completion of the combined consolidation chemotherapy, the antibody may be administered in one or more further cycles of antibody treatment. Optionally, the antibody may also be administered in one or more further cycles of antibody treatment after completion of the combined induction chemotherapy but before the combined consolidation chemotherapy.

[0282] In one embodiment, the subject has a newly diagnosed relapsed GD2-positive Ewing sarcoma. In an alternative embodiment, the subject has relapsed GD2-positive Ewing sarcoma. In a further embodiment, the subject has an initial relapsed Ewing sarcoma. In further embodiments, the subject has a second or subsequent relapsed Ewing sarcoma.

[0283] In one embodiment, the subject has a newly diagnosed high-risk metastatic GD2- positive Ewing sarcoma. In an alternative embodiment, the subject high-risk metastatic GD2-positive Ewing sarcoma. Dosage regimen

[0284] In the third aspect of the invention, there is provided a method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof.

[0285] Use or treatments described in relation to the first and second aspects can be implemented in respect to the third aspect. All features disclosed in relation to the first and second aspects are applicable to the third aspect, mutatis mutandis.

[0286] In the context of this invention, "combination chemotherapy" (such as "combination induction chemotherapy" and / or "combination consolidation chemotherapy") means that both the anti-GD2 antibody and chemotherapy (such as induction chemotherapy and / or consolidation chemotherapy) are administered in combination as a treatment to the patient, which may be referred to as a combination (chemo)therapy. By "in combination" or "combination therapy" we do not necessarily mean that the antibody dose is administered at the same time as a chemotherapy dose. By "combinations thereof", we refer to the use of more than one chemotherapy treatment being in combination with an anti-GD2 antibody.

[0287] In one embodiment, the treatment comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti- GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as an induction chemotherapy.

[0288] In some embodiments, the treatment comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as a consolidation chemotherapy. In some embodiments, the treatment comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as a maintenance chemotherapy.

[0289] In some embodiments, the treatment comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as a chemotherapy regimen.

[0290] In some embodiments, the treatment comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as a salvage chemotherapy regimen.

[0291] The present invention provides for the use of two or more chemotherapy phases, for example, 2, 3, or 4 chemotherapy phases. Additional medical interventions may take place during or between the two or more chemotherapy phases, for example, surgery, radiation, and / or non-anti-GD2 immunotherapy.

[0292] In some embodiments, the two or more chemotherapy phases are administered to the subject consecutively. For example, the subject may receive induction chemotherapy and consolidation chemotherapy. For example, the subject may receive induction chemotherapy, consolidation chemotherapy and maintenance chemotherapy. For example, the subject may receive induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy and non-standard chemotherapy.

[0293] In treatment regimens with two or more chemotherapy phases, an anti-GD2 antibody may be administered during all phases. Alternatively, an anti-GD2 antibody may not be administered to all phases. For example, the anti-GD2 antibody treatment may be discontinued in situations wherein the subject has a high risk of developing TLS or CRS.

[0294] Accordingly, in some embodiments, an anti-GD2 antibody is administered as a part of a combination chemotherapy in some chemotherapy phases of a treatment regimen. For example, the anti-GD2 antibody may be administered in the induction and consolidation phases but not in the maintenance phase. As another example, the anti- GD2 antibody may be administered in the induction phase but not in the consolidation phase and optionally not in the maintenance phase.

[0295] Additionally, in some instances it may be preferable to continue anti-GD2 antibody immunotherapy after completion of one or more phases of combination chemotherapy. Consequently, in one embodiment, the treatment can comprise the administration of an anti-GD2 antibody in combination with one or more chemotherapy phases, wherein the anti-GD2 antibody immunotherapy is maintained after completion of the one or more phases. This may be advantageous as a prophylactic measure to prevent re- emergence of the disease or to reduce the risk of early relapse.

[0296] Individual doses and dose ranges of the anti-GD2 antibody presented anywhere herein may be applied to the third aspect of the invention, provided that they are within the overall range of from 4-8 mg / m2 / d. In one embodiment, the anti-GD2 antibody is administered at a dose of from about 5 mg / m2 / d to about 7.5 mg / m2 / d, optionally at a dose of 5 mg / m2 / d or at a dose of 7.5 mg / m2 / d.

[0297] Accordingly, the dose of anti-GD2 antibody administered to the subject can be selected from a range of about 5 mg / m2 / d to about 7.5 mg / m2 / d. For example, the dose of anti- GD2 antibody administered to the subject can be 5, 5.5, 6, 6.5, 7 or 7.5 mg / m2 / d. In some instances, the dose of anti-GD2 antibody administered to the subject is maintained across each treatment cycle. In other instances, the dose of anti-GD2 antibody administered to the subject is varied between treatment cycles. In further instances, the dose of anti-GD2 antibody administered to the subject is gradually lowered in each subsequent dose. Conversely, the dose of anti-GD2 antibody administered to the subject is gradually increased in each subsequent dose. Independent doses in each phase of treatment may be varied in relation to the subject's tolerance and to reduce the risk of CSR or TLS. The skilled person would recognise the appropriate clinical indicators and increase or decrease the dose accordingly.

[0298] In an embodiment, prior to the combination therapy in accordance with the third aspect of the invention, the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, as described above.

[0299] In an additional embodiment according to the third aspect of the invention, the antibody may be further administered in one or more antibody treatment cycles without chemotherapy. In one embodiment, the subject has a newly diagnosed relapsed GD2-positive Ewing sarcoma. In an alternative embodiment, the subject has relapsed GD2-positive Ewing sarcoma. In a further embodiment, the subject has an initial relapsed Ewing sarcoma. In further embodiments, the subject has a second or subsequent relapsed Ewing sarcoma.

[0300] In one embodiment, the subject has a newly diagnosed high-risk metastatic GD2- positive Ewing sarcoma. In an alternative embodiment, the subject has high-risk metastatic GD2-positive Ewing sarcoma.

[0301] Treatment of relapsed GD2-positive Ewing sarcoma

[0302] In the fourth aspect of the invention, there is provided a method of treating relapsed GD2-positive Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma, in a subject in need thereof, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof.

[0303] Use or treatments described in relation to the first, second or third aspects can be implemented in respect to the fourth aspect. All features disclosed in relation to the first, second and third aspects are applicable to the fourth aspect, mutatis mutandis.

[0304] In some embodiments, the treatment of relapsed GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as induction chemotherapy.

[0305] In some embodiments, the treatment of relapsed GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as consolidation chemotherapy. In some embodiments, the treatment of relapsed GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as maintenance chemotherapy.

[0306] In some embodiments, the treatment of relapsed GD2-positive Ewing sarcoma in a subject in need thereof comprises a combination chemotherapy comprising (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s) as salvage chemotherapy.

[0307] The term "relapsed" includes the definition as described in connection with the first aspect of the invention.

[0308] In the context of the present application, the starting point for defining the onset of relapse can be:

[0309] (a) a remission diagnosis;

[0310] (b) the completion of the previous therapy regimen; and / or

[0311] (c) commencement of maintenance therapy.

[0312] Relapsed disease can be broadly categorised as "early" or "late". The former, arising in less than 6 months from any of (a)-(c), is typically considered to be more aggressive. The latter, which occurs 6 or more months from any of (a)-(c), is considered less aggressive.

[0313] Accordingly, in one embodiment, the subject in need of treatment has relapsed Ewing sarcoma that was detected 6 or more months after:

[0314] (a) a remission diagnosis;

[0315] (b) the completion of the previous therapy regimen; and / or

[0316] (c) commencement of maintenance therapy.

[0317] The combination therapies described herein can be used to treat early and late relapsed disease.

[0318] In an embodiment, the subject has late relapsed Ewing sarcoma; wherein the subject has relapsed after 6 or more months after:

[0319] (a) a remission diagnosis;

[0320] (b) the completion of the previous therapy regimen; and / or (c) commencement of maintenance therapy.

[0321] For example, the subject has relapsed after 6, 9, 12, 15, or 18 months or more from any of (a)-(c).

[0322] In an alternative embodiment, the subject has early relapsed Ewing sarcoma; wherein the subject has relapsed after less than 6 months after any of (a)-(c). For example, the subject has relapsed after 5, 4, 3, 2 or 1 month(s) from any of (a)-(c).

[0323] In one embodiment, the subject has had no more than a single prior line of therapy to treat an indicator selected from GD2-positive cancer, Ewing sarcoma or GD2-positive Ewing sarcoma.

[0324] In an embodiment, prior to the combination therapy in accordance with the fourth aspect of the invention, the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, as described above.

[0325] In an additional embodiment according to the fourth aspect of the invention, the antibody may be further administered in one or more antibody treatment cycles without chemotherapy.

[0326] Certain embodiments of the first aspect of the invention may be particularly useful in treating relapsed GD2-positive Ewing sarcoma. This may include, for example, embodiments or non-limited examples relating to Temozolomide & Irinotecan (TEMIRI), Topotecan & Cyclophosphamide (TOPOCYLO), or high dose Ifosfamide (HD IFOS).

[0327] Treatment of high-risk metastatic GD2-positive Ewing sarcoma

[0328] In the fifth aspect of the invention, there is provided a method of treating high-risk metastatic GD2-positive Ewing sarcoma, particularly newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma, in a subject wherein at least one metastatic tumoral site is distally located from the lung, the method comprising a combination chemotherapy of (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s). The anti-GD2 antibody can be administered during any phase of chemotherapy, such as chemotherapy selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof. By "distally located from the lung" we mean locations outside of the lung and / or pulmonary system. This may include tissues / organs located within the thoracic cavity (chest cavity) and / or tissues / organs adjacent to the lung. Potential metastatic sites would include bone marrow, kidneys, heart, adrenal glands and other soft tissues. Consequently, this aspect would not include subjects with primary pulmonary Ewing sarcoma. The location of the primary site, and metastatic sites would be determined by the skilled person using known techniques in the art. For example, metastatic sites can be identified through MRI or using a computerised tomography scan.

[0329] In an embodiment, prior to the combination therapy in accordance with the fifth aspect of the invention, the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, as described above.

[0330] In some embodiments in accordance with any of the aspects of the invention disclosed herein, the subject does not have refractory Ewing sarcoma.

[0331] By "refractory", we mean a cancer that has not responded (primary refractory), or has stopped responding (secondary refractory), to treatment. "Primary refractory" refers to a patient who has never achieved a complete remission from previous treatments, and who was not in partial remission at the time of the last evaluation prior to commencing treatment according to the invention. Partial remission may have been achieved at some point for the primary refractory, but the patient may no longer be in partial remission. "Secondary refractory" refers to a cancer that has stopped responding to treatment (prior to commencing treatment according to the invention).

[0332] A subject may be considered to have refractory Ewing sarcoma if at least one tumour does not respond to initial treatment. In some instances, a subject may be considered to have refractory Ewing sarcoma if at least one tumour does not respond to subsequent treatment after a partial response was achieved with initial treatment.

[0333] In an additional embodiment according to the fifth aspect of the invention, the antibody may be further administered in one or more antibody treatment cycles without chemotherapy.

[0334] Use or treatments described in relation to the first, second, third or fourth aspects can be implemented in respect to the fifth aspect. All features disclosed in relation to the first, second, third and fourth aspects are applicable to the fifth aspect, mutatis mutandis. Certain embodiments of the first aspect of the invention may be particularly useful in treating high-risk metastatic GD2-positive Ewing sarcoma. This may include, for example, embodiments or non-limited examples relating to Temozolomide & Irinotecan (TEMIRI), Topotecan & Cyclophosphamide (TOPOCYLO), or high dose Ifosfamide (HD IFOS).

[0335] Numbered embodiments

[0336] The invention is further defined by the following numbered embodiments:

[0337] Embodiment 1. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy of:

[0338] (i) administration to the subject of an anti-GD2 antibody; and

[0339] (ii) administration to the subject of at least one treatment dose of induction chemotherapy with one or more chemotherapeutic agent(s).

[0340] Embodiment 2. The method of embodiment 1, wherein the antibody in the combination induction chemotherapy is administered in a first treatment dose:

[0341] (a) simultaneously with one or more of the chemotherapeutic agent(s) in the first cycle of the induction chemotherapy;

[0342] (b) as sequential administration with the one or more chemotherapeutic agent(s) in the first cycle of the induction chemotherapy;

[0343] (c) simultaneously with one or more of the chemotherapeutic agent(s) in a second or subsequent cycle of the induction chemotherapy, such as in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle;

[0344] (d) simultaneously with one or more of the chemotherapeutic agent(s) in the fifth cycle of the induction chemotherapy;

[0345] (e) as sequential administration with the one or more chemotherapeutic agent(s) in the fifth cycle of the induction chemotherapy; or

[0346] (f) as sequential administration with the one or more chemotherapeutic agent(s) in the second or subsequent cycle of the induction chemotherapy, such as in the second, third, fourth, fifth, sixth, seventh, eighth, ninth or subsequent cycle.

[0347] Embodiment 3. The method according to embodiment 2, wherein the antibody in the combination induction chemotherapy is administered: (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy;

[0348] (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy;

[0349] (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy; or

[0350] (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the induction chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination induction chemotherapy.

[0351] Embodiment 4. The method according to any one of embodiments 1-3, wherein prior to the combination chemotherapy the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, optionally wherein the subject has at least one confirmed GD2 positive tumour, biopsy or cell sample; further optionally wherein the step of determining or confirming that the subject has GD2- positive Ewing sarcoma comprises immunostaining, flow-cytometry, RT-PCR, and / or an ELISA.

[0352] Embodiment 5. The method according to any one of embodiments 1-4, wherein the subject has relapsed or high-risk metastatic Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma or newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma.

[0353] Embodiment 6. The method according to embodiment 5, wherein the subject has relapsed Ewing sarcoma; optionally, wherein the subject has relapsed 6 or more months after:

[0354] (i) a remission diagnosis;

[0355] (ii) the completion of the previous therapy regimen; and / or

[0356] (iii) commencement of maintenance therapy.

[0357] Embodiment 7. The method according to embodiment 5, wherein the subject has high- risk metastatic Ewing sarcoma, optionally, wherein the cancer has metastasised from the bone or soft tissue to at least one other site within the body. Embodiment 8. The method according to any one of embodiments 1-7, wherein the subject has not previously received treatment with an anti-GD2 antibody for the treatment of Ewing sarcoma; optionally wherein the subject has not previously been treated with an anti-GD2 antibody for any purpose.

[0358] Embodiment 9. The method according to any of embodiments 1-8, wherein the chemotherapeutic agent is selected from one or more of the following: vincristine, ifosfamide, cyclophosphamide, doxorubicin, etoposide, temozolomide, irinotecan, and topotecan; optionally wherein:

[0359] (a) vincristine is administered at a dose of at least 2 mg / m2;

[0360] (b) ifosfamide is administered at a dose of at least 1800 mg / m2 / d in 1 hr IV infusions over 5 days;

[0361] (c) cyclophosphamide is administered at a dose of at least 1200 mg / m2;

[0362] (d) doxorubicin is administered at a dose of at least 37.5 mg / m2 / d for 24 hours;

[0363] (e) etoposide is administered at a dose of at least 100 mg / m2in 1 hr IV infusions over 5 days;

[0364] (f) temozolomide is administered at a dose of at least 75 mg / m2;

[0365] (g) irinotecan is administered at a dose of at least 125 mg / m2; and / or

[0366] (h) topotecan is administered at a dose of at least 1.5 mg / m2.

[0367] Embodiment 10. The method according to any one of embodiments 1-9, wherein at least 2, 3, or 4 chemotherapeutic agents are administered to the subject as part of the induction chemotherapy.

[0368] Embodiment 11. The method according to embodiment 10, wherein 5 chemotherapeutic agents are administered to the subject as part of the induction chemotherapy.

[0369] Embodiment 12. The method according to any one of embodiments 1-11, wherein the chemotherapeutic agents administered to the patient are in combinations selected from:

[0370] (i) vincristine, doxorubicin, and cyclophosphamide;

[0371] (ii) ifosfamide, and etoposide;

[0372] (iii) vincristine, ifosfamide, doxorubicin, and etoposide;

[0373] (iv) vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide;

[0374] (v) temozolomide and irinotecan; and / or

[0375] (vi) topotecan and cyclophosphamide. Embodiment 13. The method according to embodiment 12, wherein two or more chemotherapeutic agent combinations are administered in alternating cycles of induction chemotherapy.

[0376] Embodiment 14. The method according to embodiment 13, wherein at least two cycles of induction chemotherapy are administered; optionally, wherein at least 5 cycles of induction chemotherapy are administered; further optionally, wherein at least 9 cycles of induction chemotherapy are administered.

[0377] Embodiment 15. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is administered at a dose of from about 1 mg / m2 / d to about 20 mg / m2 / d; optionally wherein the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d to about 10 mg / m2 / d, such as at a dose from about 5 mg / m2 / d to about 10 mg / m2 / d or at a dose from about 4 mg / m2 / d to about 8 mg / m2 / d.

[0378] Embodiment 16. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody dose is selected from 1 mg / m2 / d to about 20 mg / m2 / d according to the subject's tolerance.

[0379] Embodiment 17. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is administered at a dose of about 10 mg / m2 / d.

[0380] Embodiment 18. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is administered at a dose of about 7.5 mg / m2 / d.

[0381] Embodiment 19. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is administered at a dose of about 5 mg / m2 / d.

[0382] Embodiment 20. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is administered as a continuous infusion over 24 hours and over consecutive days until the dose for the cycle has been administered in full.

[0383] Embodiment 21. The method according to any of the preceding embodiments, wherein the anti-GD2 antibody is a chimeric anti-GD2 antibody; optionally, wherein the anti- GD2 antibody is Dinutuximab or Dinutuximab beta; further optionally, wherein the antibody has the heavy chain sequence SEQ ID NO: 19 and the light chain sequence SEQ ID NO: 18. Embodiment 22. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy according to any one of embodiments 1-21, followed by a combination consolidation chemotherapy of:

[0384] (i) administration to the subject of an anti-GD2 antibody; and

[0385] (ii) administration to the subject of at least one treatment dose of consolidation chemotherapy with one or more chemotherapeutic agent(s).

[0386] Embodiment 23. The method of embodiment 22, wherein the antibody in the combination consolidation chemotherapy is administered in a first treatment dose:

[0387] (a) simultaneously with one or more of the chemotherapeutic agent(s) in the first cycle of the consolidation chemotherapy;

[0388] (b) as sequential administration with the one or more chemotherapeutic agent(s) in the first cycle of the consolidation chemotherapy;

[0389] (c) simultaneously with one or more of the chemotherapeutic agent(s) in a second or subsequent cycle of the consolidation chemotherapy, such as in a second, third, fourth, fifth, sixth, or subsequent cycle; or

[0390] (d) as sequential administration with the one or more chemotherapeutic agent(s) in a second or subsequent cycle of the consolidation chemotherapy, such as in the second, third, fourth, fifth, sixth, or subsequent cycle.

[0391] Embodiment 24. The method according to embodiment 23, wherein the antibody in the combination consolidation chemotherapy is administered:

[0392] (a) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy;

[0393] (b) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy;

[0394] (c) simultaneously with one or more of the chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy; or

[0395] (d) as sequential administration with the one or more chemotherapeutic agent(s) in each cycle of the consolidation chemotherapy subsequent to the cycle in which the first treatment dose of the antibody is given in the combination consolidation chemotherapy. Embodiment 25. The method according to any one of embodiments 22-24, wherein the one or more chemotherapeutic agents in the consolidation chemotherapy is selected from vincristine, ifosfamide, cyclophosphamide, doxorubicin, etoposide, temozolomide, irinotecan, and topotecan; and wherein combinations are selected from:

[0396] (i) vincristine, doxorubicin, and cyclophosphamide;

[0397] (ii) ifosfamide, and etoposide;

[0398] (iii) vincristine, ifosfamide, doxorubicin, and etoposide;

[0399] (iv) vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide;

[0400] (v) temozolomide and irinotecan; and / or

[0401] (vi) topotecan and cyclophosphamide.

[0402] Embodiment 26. The method according to embodiment 25, wherein two or more chemotherapeutic agent combinations are administered in alternating cycles of consolidation chemotherapy.

[0403] Embodiment 27. The method according to embodiment 26, wherein at least two cycles of consolidation chemotherapy are administered; optionally, wherein at least 3 cycles of consolidation chemotherapy are administered; further optionally, wherein at least 5 cycles of consolidation chemotherapy are administered.

[0404] Embodiment 28. The method according to embodiment 27, wherein the anti-GD2 antibody is administered in the combination consolidation chemotherapy at a dose that is:

[0405] (i) lower than a dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy; or

[0406] (ii) at a dose that is 75% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy;

[0407] (iii) at a dose that is 50% of the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy; or

[0408] (iv) the same as the dose at which the anti-GD2 antibody is administered in the combination induction chemotherapy.

[0409] Embodiment 29. A method of treating relapsed GD2-positive Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma, in a subject in need thereof, the method comprising a combination chemotherapy of:

[0410] (i) administration to the subject of an anti-GD2 antibody; and (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof.

[0411] Embodiment 30. The method according to embodiment 29, wherein the Ewing sarcoma has relapsed after 6 or more months after:

[0412] (a) a remission diagnosis;

[0413] (b) the completion of the previous therapy regimen; and / or

[0414] (c) commencement of maintenance therapy.

[0415] Embodiment 31. The method according to embodiment 29 or 30, wherein prior to the combination chemotherapy the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, optionally wherein the subject has at least one confirmed GD2 positive tumour, biopsy or cell sample; further optionally wherein the step of determining or confirming that the subject has GD2- positive Ewing sarcoma comprises immunostaining, flow-cytometry, RT-PCR, and / or an ELISA.

[0416] Embodiment 32. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination chemotherapy of:

[0417] (i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and

[0418] (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy and combinations thereof.

[0419] Embodiment 33. The method according to embodiment 32, wherein the anti-GD2 antibody is administered at a dose of from about 5 mg / m2 / d to about 7.5 mg / m2 / d, optionally at a dose of 5 mg / m2 / d or at a dose of 7.5 mg / m2 / d.

[0420] Embodiment 34. The method according to embodiment 32 or 33, wherein prior to the combination chemotherapy the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, optionally wherein the subject has at least one confirmed GD2 positive tumour, biopsy or cell sample; further optionally wherein the step of determining or confirming that the subject has GD2- positive Ewing sarcoma comprises immunostaining, flow-cytometry, RT-PCR, and / or an ELISA.

[0421] Embodiment 35. A method of treating high-risk metastatic GD2-positive Ewing sarcoma, particularly newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma, in a subject wherein at least one metastatic tumoral site is distally located from the lung, the method comprising a combination chemotherapy of:

[0422] (i) administration to the subject of an anti-GD2 antibody; and

[0423] (ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or combinations thereof.

[0424] Embodiment 36. The method according to embodiment 35, wherein prior to the combination chemotherapy the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, optionally wherein the subject has at least one confirmed GD2 positive tumour, biopsy or cell sample; further optionally wherein the step of determining or confirming that the subject has GD2- positive Ewing sarcoma comprises immunostaining, flow-cytometry, RT-PCR, and / or an ELISA.

[0425] Embodiment 37. The method according to any previous numbered embodiment, wherein the subject does not have refractory Ewing sarcoma.

[0426] Embodiment 38. The method according to any previous numbered embodiment, wherein the method further comprises administration of the anti-GD2 antibody to the subject in one or more antibody treatment cycles without chemotherapy.

[0427] Embodiment 39. The method according to any one of embodiments 1-38, wherein the chemotherapeutic agent is temozolomide and irinotecan (TEMIRI) or is cyclophosphamide and topotecan (TOPOCYCLO), and wherein the anti-GD2 antibody is dinutuximab beta.

[0428] Embodiment 40. The method according to embodiment 39, wherein the dinutuximab beta is administered at a dose of 10 mg / m2 / day for 7 days per cycle.

[0429] Embodiment 41. The method according to embodiment 39, wherein the dinutuximab beta is administered at a dose of 10 mg / m2 / day for 10 days per cycle. Embodiment 42. The method according to any one of embodiments 39-41, wherein the combination induction chemotherapy is administered for 6-12 cycles of 21-28 days per cycle.

[0430] Embodiment 43. The method according to embodiment 42, wherein the combination induction chemotherapy is administered for 6 cycles of 21-28 days per cycle.

[0431] Embodiment 44. The method according to any one of embodiments 1-38, wherein the chemotherapeutic agent is ifosfamide, and wherein the anti-GD2 antibody is dinutuximab beta.

[0432] Embodiment 45. The method according to embodiment 44, wherein the ifosfamide is administered at a dose of at least 3 g / m2 / d for 5 days.

[0433] Embodiment 46. The method according to embodiment 44 or 45, wherein the dinutuximab beta is administered at a dose of 10 mg / m2 / day for 7 days per cycle.

[0434] Embodiment 47. The method according to any one of embodiments 44-46, wherein the dinutuximab beta is administered at a dose of 10 mg / m2 / day for 10 days per cycle.

[0435] Embodiment 48. The method according to any one of embodiments 44-47, wherein the combination induction chemotherapy is administered for 4 cycles of 21-28 days per cycle.

[0436] DESCRIPTION OF THE FIGURES

[0437] Figure 1. Dinutuximab beta induces ADCC in Ewing's sarcoma cells in vitro a) FACS analysis on TC-71 either stained or not with Dinutuximab beta conjugated to IRDye800CW fluorophore. b) Cell viability assay. TC-71 cells were treated with either Dinutuximab beta or Doxorubicin at the indicated concentrations for 24 or 48 hours. Cell viability is shown as % of viable cells relative to the control group (vehicle treated), c) Antibody-dependent cellular cytotoxicity was evaluated with the ADCC Reporter Bioassay. TC-71 cells were treated with Dinutuximab beta at the indicated concentrations. Data are expressed as arbitrary value of signal intensity, d) (Left panel) Confocal images of TC-71 cells stained, without permeabilization, for GD2 and plasma membrane. Nuclei were counterstained with DAPI. (middle and right panel) Magnification of the cropped area. Arrows indicate GD2 expressed on the plasma membrane. Scale bar = 10pm. e) Antibody-dependent cellular cytotoxicity was evaluated with the ADCC Reporter Bioassay. TC-71 cells were treated with either Dinutuximab beta or IgG isotype, at the indicated concentrations. As negative control, GD2-negative CHO-K1 cells were treated with Dinutuximab beta at the same concentrations. Data are expressed as arbitrary value of signal intensity, f) GD2 surface expression (ABC = antibodies bound per cell) as measured by flow cytometry and quantified with BD Quantibrite beads. Data are presented as mean ± S.E.M. , p< 0.05 (Student's t-test)

[0438] Figure 2. Dinutuximab beta inhibits tumour growth and increase survival in vivo a) Diagram of the in vivo experiment. The diagram indicates the amount and the days the various drugs were administered to the animals as well as the two time points at which the tumours of the satellite groups were collected, b) Tumour growth curves obtained in TC-71 tumour-bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Tumour size is expressed as mg. Complete statistical analysis is reported in Table 8. c) Survival curves obtained in TC-71 tumour-bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Complete statistical analysis is reported in Table 9.

[0439] Figure 3. Dinutuximab beta induces partial tumour shrinkage a) Growth curves of individual tumours. Tumour size is expressed as mg. b) Change in tumour size for each tumour at day 4 compared to day 0. Each bar represents the size of one tumour. Data are shown as the difference between tumour size at day 4 (in mg) and tumour size at day 0 (in mg). In the Dinutuximab beta, Doxorubicin and Combination group, 3 out of 9, 3 out of 9 and 5 out of 9 tumours decreased in size, respectively, c) Plot showing average body weight of TC-71 tumour bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Data are shown in grams.

[0440] Figure 4. Dinutuximab beta inhibits tumour cell proliferation and induces necrosis a) Representative pictures of tumours stained by Haematoxylin and Eosin (H&E), collected at day 4 (Tl) and day 11 (T2) after treatment start, b) Histopathological evaluation of necrotic tissue expressed as % of necrotic tissues compared to total tumour tissue. T1 and T2, p < 0.05 among groups (Kruskal-Wallis test), * p < 0.05 vs Vehicle (Uncorrected Dunn's test). c) Histopathological count of mitotic cells per field. Tl, p=0.122 among groups (Kruskal-Wallis test); T2, p < 0.05 among groups (Kruskal-Wallis test), * p < 0.05 vs Vehicle (Uncorrected Dunn's test), d) Histopathological count of apoptotic cells per field. Tl and T2, p < 0.01 among groups (Kruskal-Wallis test), * p < 0.05 vs Vehicle (Uncorrected Dunn's test), e) Graphical representation of the predominant localization of necrosis within the tumor. Data are presented as mean ± S.E.M. A score was assigned depending on the primary distribution pattern of necrosis. (See methods section for a description of the scoring system).

[0441] Figure 5. Dinutuximab beta has a short half-life in tumour-bearing mice a-b) Pharmacokinetic profile of Dinutuximab beta administered as single dose of 15mg / kg intraperitoneally in mice, a) Plot of the plasma concentration of Dinutuximab beta over time and b) pharmacokinetic parameters, c) Plasma concentration of Dinutuximab beta in blood samples from the animals of the satellite groups treated with Dinutuximab beta; four sample per each time point.

[0442] Figure 6. A two-cycle regimen enhances the effectiveness of combination therapy a) Diagram of the in vivo experiment. The diagram indicates the amount and the days the various drugs were administered to the animals as well as the time point at which the tumours of the satellite groups were collected, b) Tumour growth curves obtained in TC-71 tumour bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Tumour size is expressed as mg. Complete statistical analysis is reported in Table 10. c) Survival curves obtained in TC-71 tumour bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Complete statistical analysis is reported in Table 11.

[0443] Figure 7. Two cycles of combination therapy induce tumour shrinkage a) Growth curves of individual tumours. Tumour size is expressed as mg. b) Change in tumour size for each tumour at day 5 and day 12 compared to day 0. For the Sequential Combination group, tumour size at day 7 and 17 are shown. Each bar represents the size of one tumour. Data are shown as the difference between tumour size at the indicated day (in mg) and tumour size at day 0 (in mg), c) Plot showing average body weight of TC-71 tumour bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Data are shown in grams.

[0444] Figure 8. Protocol overview for the phase I trial of dinutuximab beta and vincristine / doxorubicin / cyclophosphamide and ifosfamide / etoposide in children, adolescents, and in GD2 positive Ewing sarcoma as described in Example 2.

[0445] Figure 9. Protocol overview for a proposed phase II trial of dinutuximab beta and chemotherapy with TEMIRI or TOPOCYCLO as described in Example 3. Abbreviations: TEMIRI: Temozolomide & Irinotecan; TOPOCYCLO: Cyclophosphamide & Topotecan; SOC: Standard of Care; RT: Radiotherapy; EFS: Event Free Survival; OS: Overall Survival; ORR: Overall Response Rate; DOR: Duration of Response; PRO: patients' reported outcome questionnaire; HRQoL: Health-related quality of life, and FU: followup

[0446] Figure 10. Protocol overview for a proposed treatment schedule for the proposed phase II trial of dinutuximab beta and chemotherapy with TEMIRI (Temozolomide & Irinotecan) or TOPOCYCLO (Cyclophosphamide & Topotecan) as described in Example 3.

[0447] Figure 11. Protocol overview for a proposed treatment schedule for a proposed phase II trial of dinutuximab beta and chemotherapy with HD-IFOS (High-dose Ifosfamide) as described in Example 3.

[0448] Figure 12. Spatial distribution of necrosis.

[0449] Representative H&E images showing the distribution of necrosis across tumor specimen under different conditions. The histological evaluation is reported in methods section.

[0450] Figure 13. Sequencing metrics and Principal component analysis

[0451] (a-b) Bar plot showing reads length for each sample before (a) and after (b) filtering. T1 = time point 1; T2 = time point 2; CTR = vehicle; QA = Dinutuximab beta. Samples T1_CTR_1 and T2_QA_1 display reduced reads length, likely due to partial degradation, c-d) Bar plot showing the percentage of alignments on human (c) and murine (d) genome for each sample, e-f) Principal component analysis showing the distribution of each sample for the human (e) and murine (f) genome. Samples T1_CTR_1 and T2_QA_1 exhibited strong separation from their respective experimental groups in the PCA space, indicating potential technical anomalies. Based on their anomalies both in the reads length and PCA analysis, these samples were therefore excluded from downstream differential expression analyses to ensure robust comparisons between groups.

[0452] Figure 14. RNAseq analysis revealed infiltration of immune effector cells into Dinutuximab-treated tumors

[0453] Differential Expression and Gene Ontology analysis of murine transcripts a) Heatmap showing the differentially expressed transcripts between Vehicle and Dinutuximab treated groups atTl. Transcripts with adjusted p-value < 0.05 are shown. b) Gene Ontology analysis of the protein-coding mRNA differentially expressed between Vehicle and Dinutuximab treated groups at Tl. The Bar plot shows the top 20 significantly enriched biological process GO terms (p-value < 0.05). c) Heatmap showing the differentially expressed transcripts between Vehicle and Dinutuximab treated groups atT2. Transcripts with adjusted p-value < 0.05 are shown. d) Gene Ontology analysis of the protein-coding mRNA differentially expressed between Vehicle and Dinutuximab treated groups at T2. The Bar plot shows the top 20 significantly enriched biological process GO terms (p-value < 0.05).

[0454] Figure 15. Analysis on murine genome. a, c) Principal component analysis at time points 1 (a) and 2 (c). CTR = vehicle; QA = Dinutuximab beta, b, d) Volcano plots showing differentially expressed transcripts from Dinutuximab beta and vehicle treated tumors at Tl (b) and T2 (d). CTR = vehicle; QA = Dinutuximab beta. Only results with an adjusted p-value <0.05 are visualized.

[0455] Figure 16. RNAseq analysis revealed biological processes involved in cell cycle arrests and cell death in tumors treated with Dinutuximab

[0456] Differential Expression and Gene Ontology analysis of human transcripts a) Heatmap showing the differentially expressed transcripts between Vehicle and Dinutuximab treated groups atT2. Transcripts with adjusted p-value < 0.05 are shown. b) Gene Ontology analysis of the protein-coding mRNA differentially expressed between Vehicle and Dinutuximab treated groups at T2. The Bar plot shows the top 20 significantly enriched biological process GO terms (p-value < 0.05). Figure 17. Analysis on human genome. a, c) Principal component analysis at time points 1 (a) and 2 (c). CTR = vehicle; QA = Dinutuximab beta, b, d) Volcano plots showing differentially expressed transcripts from Dinutuximab beta and vehicle treated tumors at T1 (b) and T2 (d). CTR = vehicle; QA = Dinutuximab beta. Only results with an adjusted p-value <0.05 are visualized.

[0457] Figure 18. Treatment scheme described in Example 4.

[0458] Diagram showing the treatment scheme. The diagram indicates the days the various drugs were administered to the animals. Doses are reported in Table 12.

[0459] Figure 19. Dinutuximab beta in combination with chemotherapy inhibits tumor growth and extends mice survival. a) Tumor growth curves obtained in TC-71 tumor bearing mice treated as indicated in Figure 18. Tumor size is expressed as mm2. Complete statistical analysis is reported in Table 13. b) Survival curves obtained in TC-71 tumor bearing mice treated, c) Change in tumor size for each tumor at day 11 compared to day 0. Each bar represents the size of one tumor. Data are shown as the difference between tumor size at day 4 (in mm2) and tumor size at day 0 (in mm2), d) Plot showing average body weight of TC- 71 tumor bearing mice treated with Dinutuximab beta, doxorubicin and their combination. Data are shown in grams.

[0460] EXAMPLES

[0461] The invention will now be illustrated by the following non-limiting examples.

[0462] Example 1: A pre-clinical in vitro and murine study demonstrating the efficacy of dinutuximab beta in combination with the chemotherapeutic agent doxorubicin for the treatment of Ewing sarcoma.

[0463] Example 2: Phase I trial of dinutuximab beta and vincristine / doxorubicin / cyclophosphamide and ifosfamide / etoposide in children, adolescents, and in GD2 positive Ewing sarcoma.

[0464] Example 3: A proposed multicentre, international, randomized, parallel, open label, phase II trial evaluating the efficacy and safety of the combination of dinutuximab beta with standard chemotherapy in Ewing Sarcoma relapsed patients. In the context of this example, standard chemotherapy relates to chemotherapy in accordance with the first, second, and fourth aspects of the invention.

[0465] Example 4: Pre-clinical evaluation of the efficacy of Dinutuximab beta in addition to the standard chemotherapy backbone against Ewing Sarcoma.

[0466] Example 5: A proposed multicentre, international, randomized, open label Phase III trial evaluating the efficacy of dinutuximab beta in combination with irinotecan and temozolomide in Ewing Sarcoma relapsed patients.

[0467] Example 1

[0468] 1. Pre-clinical in vitro and murine study demonstrating the effect of dinutuximab beta in combination with doxorubicin for the treatment of Ewing's sarcoma

[0469] References to Dinutuximab in the following example and associated figures should be taken to mean "Dinutuximab beta" as described in the sub-section 1.2 entitled "Materials and methods".

[0470] 1.1. Introduction and study rationale

[0471] Ewing's sarcoma (EwS) is a rare and aggressive form of cancer that affects the bones and soft tissues, and it is estimated that around 250 people in the United States and 600 in Europe get diagnosed with it every year. EwS mostly affects children and AYAs (adolescent and young adults) with a peak incidence at the age of 15 years, and it is slightly more common in male than females (sex ratio of 3:2). EwS is mainly seen in European descendent populations with an estimated incidence of ~1.5 cases per million children and AYAs. The estimated incidence in Asian and African populations is much lower, with annual rates of ~0.8 and ~0.2 cases per million children, respectively (Grunewald, T.G.P., et al. Nat Rev Dis Primers. 2018 Jul 5;4(1):5).

[0472] EwS can arise in any bone of the body, but most commonly in the pelvis, femur, tibia, humerus, and ribs. Ewing's sarcoma can also occur in the soft tissues, such as the muscles, nerves, fat, or blood vessels, in which case it is called extraosseous EwS. At the genetic level, the hallmark of EwS is a chromosomal rearrangement that causes the EWS gene to join with one of the various ETS transcription factors, with FLU being the most frequent partner. The fusion protein acts as an aberrant transcription factor that drives the expression of oncogenic genes and suppresses the expression of tumour suppressor genes (Ramachandran, B., et al. 2021. Am J Transl Res 13, 12181-12194; Zollner, S.K., et al. J Clin Med. 2021 Apr 14;10(8): 1685). The current standard of care for patients with EwS consists of multimodal therapy, including surgery, radiotherapy, and chemotherapy. Surgery and radiotherapy are used to remove or destroy the primary tumour and any local or regional metastases. Chemotherapy is used to eradicate any systemic or microscopic metastases, and to prevent or delay the recurrence or the progression of the disease. Chemotherapy is usually based on a combination of agents, such as vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide. However, despite the intensive treatment, the prognosis of patients with EwS remains poor, especially for those with metastatic or recurrent disease. The 5-year survival rate for patients with localized disease is around 70%, while for those with metastatic disease it drops to 20-30%. Moreover, the chemotherapy regimen is associated with significant toxicity and long-term sequelae, such as cardiotoxicity, nephrotoxicity, infertility, and secondary malignancies (Grunewald, T.G.P., et al. Nat Rev Dis Primers. 2018 Jul 5;4(1):5; Zollner, S.K., et al. J Clin Med. 2021 Apr 14;10(8): 1685). Therefore, there is an urgent need for new therapeutic strategies that can improve the outcome and the quality of life of patients with EwS, by increasing the efficacy and reducing the toxicity of the treatment. One promising approach is the combination of chemotherapy with immunotherapy, which aims to harness the immune system to recognize and eliminate the tumour cells. In order to be effective, immunotherapy requires the presence of antigens that are predominantly expressed in the tumour cells. One of these antigens is disialoganglioside GD2 which is highly express by almost all the tumours of neuroectodermal origin, such as neuroblastoma, osteosarcoma, melanoma, and small cell lung cancer. The GD2 antigen is not expressed by normal tissues, except for the peripheral nerves and the brain. This makes it an attractive target for immunotherapy, as it can distinguish the tumour cells from the normal cells (Nazha, B., et al. Front Oncol. 2020 Jul 7: 10: 1000).

[0473] Ewing's sarcoma has been studied for GD2 expression, and the results ranged from no detectable surface expression to diffused and / or intense staining in some tumours (Bailey, K., et al. FlOOORes. 2019 Apr 15:8:F1000 Faculty Rev-493). More specifically, GD2 expression levels ranged from 40% to 90% from diagnostic biopsy samples of Ewing's sarcoma (Mora, J., et al. Front Oncol. 2019 Jun 19:9:53). Kailayangiri et al., detected GD2 expression by immunofluorescence staining in 10 of 10 EwS cell lines and 3 of 3 primary cell cultures, concluding that surface expression of GD2 is characteristic of EwS and that GD2 provides an appropriate target antigen for therapeutic strategies to eradicate micro metastases and reduce the risk of recurrence in patients with high-risk disease (Kailayangiri, S., e al. Br J Cancer. 2012 Mar 13;106(6): 1123-33).

[0474] Dinutuximab beta is a monoclonal antibody that targets the GD2 antigen expressed by tumour cells. Dinutuximab beta binds to the GD2 antigen and recruits the immune effector cells, such as natural killer cells and macrophages, to mediate antibodydependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the tumour cells.

[0475] Dinutuximab beta is indicated for high-risk neuroblastoma in patients over 12 months old, who have responded at least partially to prior treatments, or who have had neuroblastoma relapsed or refractory, with or without any remaining disease. According to an anecdotal report, three patients who had newly diagnosed, metastatic, GD2-positive Ewing's sarcoma received Dinutuximab beta along with standard consolidation chemotherapy, and in addition received local therapy comprising surgery and radiation. (Spasov, N ., et al. 2022. J Pediatr Hematol Oncol. 2022 Aug I;44(6):e948-e953). However, the efficacy of Dinutuximab beta in treating Ewing's sarcoma and the underlying molecular and cellular mechanisms of its therapeutic action have not been previously investigated. Utilizing both in vitro and in vivo approaches, we provide, for the first time, evidence that Dinutuximab beta effectively suppresses Ewing's sarcoma growth, functioning both as a monotherapy and in combination with the standard chemotherapeutic agent doxorubicin.

[0476] 1.2. Materials and methods

[0477] 1.2.1. Drugs

[0478] Dinutuximab beta was provided by Recordati S.p.a . Doxorubicin (cat. HY-15142) was from MedChemExpress, the human IgGl isotype control was from BioXcell (cat. BE0297). All the reagents were prepared accordingly to the relative instructions supplied together with the compounds.

[0479] 1.2.2. Cell line TC71 cell line carrying the EWS / FLU type I translocation was a kind gift of Dr. Roberta Frapolli. The cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% Fetal Bovine serum, 2 mM glutamine, and 1% Penicillinstreptomycin. CHO-K1 cells were purchased from ATCC and maintained in a 1 : 1 mixture Minimum Essential Medium and Ham's F12 Medium supplemented with 10%FBS and 1 % Penicillin-Streptomycin. SK-N-BE(2) neuroblastoma cells were purchased from ATCC and cultured in a 1 : 1 mixture of Eagle's Minimum Essential Medium and F12 Medium supplemented with 10% Fetal Bovine serum and 1 % Penicillin-Streptomycin.

[0480] 1.2.3. Drug treatment and cell viability assay

[0481] Cell viability was assessed using the CellTiter-Glo Luminescent Viability Assay KIT (Promega, Madison, WI, USA). TC71 cells, at 80% confluence, were washed with PBS, trypsinized and then seeded in 96-well plates at the density of 15,000 cells per well in complete medium (Iscove's modified Dulbecco's medium with 10% Fetal bovine serum and 1% Penicillin / Streptomycin) and incubated at 37°C with 5% CO2. After 24 hours, the cells were treated with serial 1:3 dilution of Dinutuximab beta (2 mg / ml; starting from 4.3 pM), doxorubicin (0.2 % DMSO; starting from 300 pM) in basal medium. After 24 and 48 hours of incubation, an equal volume of CellTiter-Glo reagent to the volume of cell culture medium present in each well was added, and the plate was incubated for 10 minutes to stabilize the luminescent signal. The luminescence was then measured using the FlexStation® 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Relative cell viability was calculated as follows: cell viability at the given concentration / cell viability of control x 100. The experiment was conducted three times, each with three technical replicates.

[0482] 1.2.4. Fluorescent-activated cell sorting (FACS)

[0483] 106TC-71 cells were resuspended in lOOul of PBS + 2% FBS + 2mM EDTA and then incubated for 30 minutes at 4°C with Dinutuximab beta conjugated with the fluorophore IRDye800CW. After three washes the cells were fixed with 1% PFA and then acquired on a Cytek Aurora cell sorter. Negative controls were TC-71 cells unstained and GD2-negative cells.

[0484] 1.2.5. ABC (antibodies bound per cell) analysis SK-N-BE(2) and TC 71 cells were grown to 90% confluency and detached using trypsin. IxlO6cells / ml per tube were washed in cold FACS buffer (PBS supplemented with 2% heat-inactivated FBS and 2 mM EDTA) followed by incubation with anti-GD2-PE antibody (final concentration: 3 pg / ml; Clone 14G2a, Isotype: mouse IgG2a, BD Biosciences) or isotype control mouse IgG2a-PE,k (Isotype: control mouse IgG2a-PE,k ; BD Biosciences) for 45 minutes at 4 °C, protected from light. Cells were then washed twice with FACS buffer and transferred to filter top tubes with addition of DAPI live / dead stain (final concentration: 1 pg / ml; Sigma-Aldrich). Data were acquired using a Cytek Aurora flow cytometer, collecting 20,000 events from the live (DAPI-negative) singlet gate for each cell line. BD PE QuantiBrite Beads (BD Biosciences) were analyzed according to the manufacturer's protocol during each run. A standard curve was generated in GraphPad Prism, from which the PE geometric mean for each sample was converted into the number of antibodies bound per cell (ABC), representing surface antigen expression. Mean and S.E.M. were calculated from a minimum of three replicate values for each cell line.

[0485] 1.2.6. Immunofluorescence and confocal imaging

[0486] TC-71 cells were fixed with 4% Paraformaldehyde and then incubated with Rhodamine Red-X conjugated Dinutuximab beta for 2 hours at room temperature, without permeabilization. Cells were stained with CellMask Deep Red Plasma Membrane Stain (ThermoFisher) for 10 minutes, washed with PBS, fixed in 4% paraformaldehyde for 10 minutes at room temperature, and mounted with DAPI-containing medium for nuclei counterstain. Images were acquired with a 1X81 confocal (Olympus, Tokyo, Japan).

[0487] 1.2.7. ADCC

[0488] Antibody-dependent cellular cytotoxicity was evaluated with the ADCC Reporter Bioassay, Core Kit, from Promega (Cat. G7010), following manufacturer's instructions. A ratio effector to target cells equal to 10: 1 was used. As negative controls, both IgG isotype on TC-71 cells and Dinutuximab beta on CHO-K1 (GD2 negative) cells were used.

[0489] 1.2.8. Mice

[0490] All the procedures with animals were performed in agreement with the Institutional Animal Care and Use Committee (IACUC) of Istituto di Ricerche Farmacologiche Mario Negri IRCCS (IRFMN), in compliance with the guidelines established in the Principles of Laboratory Animal Care (Directive 86 / 609 / EEC) and as approved by the Italian Ministry of Health.

[0491] Female Athymic nude mice, 7-8 weeks old, were obtained from INOTIV (Netherlands). Male CD1, 5 weeks old mice were obtained from Charles River (Italy). They were maintained under Specific Pathogen Free conditions with constant temperature and humidity and fed with a regular chow diet and water ad libitum.

[0492] 1.2.9. Tumour models

[0493] To obtain the tumours, 200 pl of cell suspension containing 5 x 106TC71 cells were injected subcutaneously into the right flanks of the mice. The growing tumour masses were measured with the aid of a Vernier caliper, and the tumour weights (1mm3=lmg) were calculated by the formula: length x (width)2 / 2.

[0494] 1.2.10. Antitumour activity study in xenograft models

[0495] When tumour weight reached about 100 mg (about 100 mm3), mice were randomized into the treatment groups. Tumour bearing mice were monitored at least twice a week by Vernier caliper. Mice were euthanized when tumours reach a volume of > 1500 mm3, in case of tumour ulceration or body weight loss >20%. The antitumour activity was expressed as T / C%, where T and C are the mean tumour weight of treated and control groups, respectively.

[0496] Parallel groups of 4 mice for each treatment group were sacrificed ad interim at the indicated time points. Terminal blood samples (approx. 400 pl) were collected, and plasma-separated by centrifugation at 4000 rpm at 4°C for 10 minutes. Tumours were removed and immediately frozen in dry-ice or formalin fixed paraffine embedded for pharmacodynamic analysis.

[0497] 1.2.11. Pharmacokinetic study and IgG dosage

[0498] Three male, 5-week-old CD1 mice were used for the pharmacokinetic study. Mice received a single intraperitoneal injection of Dinutuximab beta at a dose of 15mg / kg. 50ul of blood were withdrawn from a small cut on the tail at 30 minutes, 6 hours, 1, 2, 5, 7 and 12 days after injection. Blood was collected in EDTA-coated tubes, kept at 4°C and centrifuged for 10 min at 4000 RPM. Plasma was immediately frozen at -20°C until the analysis. The pharmacokinetic parameters were calculated with the Phoenix WinNonlin software Version 8.4. Plasma concentration of Dinutuximab beta was determined with an ELISA kit for human IgG from AbCam (IgG Human SimpleStep ELISA® Kit, Cat. abl95215), according to manufacturer's instructions.

[0499] 1.2.12. Histology

[0500] Subcutaneous tumours were harvested and immediately fixed in 10% formalin for 24h, then put in 70% ethanol until paraffin embedding, following standard procedure. 5 pm thick sections were obtained at the microtome and stained with haematoxylin and eosin (H&E).

[0501] Histopathological evaluation: samples were examined with a light microscope for the detection of histological features of the tumour. Histopathology evaluation was made in a blind fashion, i.e. without knowledge of the treatment group. For necrosis, the percentage of affected tissue was given; moreover, the count of apoptotic and mitotic figures was made in three randomly selected microscopic fields, avoiding the areas of necrosis.

[0502] Four animals for each satellite group were analyzed. Each specimen was assigned a score depending on the primary distribution pattern of necrosis. The score system was arbitrary defined as follows:

[0503] Assigned values for the pattern of necrosis distribution:

[0504] • Central - 0

[0505] • Diffuse throughout the tumor, predominantly central - 1

[0506] • Diffuse throughout the tumor - 2

[0507] • Diffuse throughout the tumor, predominantly peripheral - 3

[0508] • Peripheral - 4

[0509] The count of apoptotic and mitotic figures was made in three randomly selected microscopy fields, avoiding the areas of necrosis.

[0510] 1.2.13. RIMA extraction

[0511] Frozen tumor sections collected at T1 and T2, were homogenized using TissueLyser LT (Qiagen, Benelux, B.V.) in Trizol Reagent (Invitrogen, Cat. No.15596026). The PureLink RNA Mini Kit (Life technologies Cat. No. 12183018A) was adopted to extract and purify total RIMA. The concentration and purity of RNA were determined at 260 / 280 nm using Nano Drop One Spectrophotometer (Thermo Fisher Scientific Inc. USA).

[0512] 1.2.14. RNA-seq Library Preparation and Sequencing

[0513] Bulk long-read RNA sequencing was performed using the Oxford Nanopore Technologies (ONT; Oxford, UK) PCR-cDNA Sequencing Kit (SQK-PCB114-24). Sequencing libraries were prepared according to the manufacturer's protocol. Briefly, 700 ng of full-length RNA per sample was used as input for complementary DNA (cDNA) synthesis and strand switching with kit-supplied oligonucleotides. Double-stranded cDNA (dscDNA) was generated by PCR amplification (10 pL cDNA / sample) using primers containing 5' tags, which enable ligase-free attachment of rapid sequencing adapters. The resulting libraries were loaded onto R10.4.1 flow cells (FLO-PRO114M) and sequenced on P2 Solo PromethlON devices using MinKNOW acquisition software (v24.ll.10).

[0514] 1.2.15. RNA-seq Raw Data Processing

[0515] Raw ONT sequencing data in POD5 format were basecalled using Dorado v7.6.8 with the high-accuracy (HAC) model v4.3.0 (400 bps) within MinKNOW (v24.11.10). Base called reads were subjected to quality control and reads filtering with fastpLong v0.3.0(Chen, 2023). Reads in which >40% of bases had a Phred score <15 were removed to ensure downstream data quality. High-quality reads were aligned to both the Gencode human transcriptome (GRCh38, v38) and the Gencode mouse transcriptome (GRCm39, vM28) using minimap2 v2.30(Li, 2018) with the map-ont preset optimized for ONT long reads. Xenomapper(Wakefield, 2016) was used to segregate human- and mouse-derived reads in xenograft samples. Transcript-level quantification was then performed using Salmon vl.l0.3(Patro et al., 2017) with parameters — gcBias — numBootstraps 10 -I U to account for GC bias and provide bootstrap estimates of variance. Transcript-level abundance estimates were aggregated to gene-level raw counts with tximport vl.32.0(Soneson et al., 2015). Batch effects were corrected using the ComBat_seq function from the sva package v3.52.0(Zhang et al., 2020). Finally, genes with low counts for a number of samples equal to the minimum group size were filtered out (< 10 counts for human, <5 counts for mouse, reflecting differences in read depth).

[0516] 1.2.16. Exploratory Analysis For exploratory analyses, raw counts were normalized with DESeq2 vl.44.0 (Love et al., 2014) and variance-stabilized using the rlog() function. Principal Component Analysis (PCA) of normalized expression values was performed with prcomp() from the stats package v4.4.1. Only results with an adjusted p-value <0.05 were retained for visualization.

[0517] 1.2.17. Differential Expression and Functional Enrichment

[0518] Differentially expressed genes (DEGs) were identified using DESeq2 vl.44.0, with significance defined as an adjusted p-value <0.05 after multiple testing correction (Benjamini-Hochberg). Functional enrichment analysis of DEG was performed using RNAenrich (Zhang et al., 2023). The Gene Ontology (GO) term Biological Process was interrogated.

[0519] 1.2.18. Statistical analysis

[0520] Tumour growth curves were analyzed using GraphPad Prism version 9 software (GraphPad software, Inc., La Jolla, CA, USA) performing generalized linear mixed- effects models as previously described (doi.org / 10.1038 / s41598-021-87470-x). This test gives the same results as repeated measures ANOVA if there are no missing values, and comparable results when there are missing values.

[0521] For the survival curves statistical analysis was performed with SAS® 9.4 software. The median and range were calculated for the survival time. A Restricted Mean Survival Time (RMST) analysis (Royston and Parmar BMC Medical Research Methodology 2013, 13: 152) comparing the area under the Kaplan-Meier survival curves (AUC), calculated up to the last experimental observation, was performed. For the data from the histopathological evaluation the Kruskal-Wallis's tests followed by the Dunn's post hoc tests was applied.

[0522] 1.3. Results

[0523] 1.3.1. Dinutuximab beta induces ADCC in Ewing's sarcoma cells in vitro

[0524] The anti-tumour activity of Dinutuximab beta was investigated in vitro in the human Ewing's sarcoma cell line TC-71. TC-71 cells carry the EWS / FLI1 type 1 translocation and are reported to express high level of GD2 (Kailayangiri, S., et al. Mol Then 2019 May 8;27(5):933-946.). Surface expression of GD2 was confirmed by FACS analysis, which showed that 88.65 ± 4.55% express high level of GD2 (Figure la). We also confirmed that TC-71 cells express GD2 at significantly lower levels compared to neuroblastoma cells (Figure If). Finally, surface expression of GD2 was confirmed by confocal imaging (Figure Id). The effect of Dinutuximab beta on TC-71 cells was then investigated in a cell viability assay. The treatment with Dinutuximab beta for up to 48h hours did not induce reduction of cell viability, even at the highest dose of 5.4 uM (Figure lb). These cells showed sensitivity to doxorubicin, with an EC50 of 1.78 uM and 0.20 uM at 24- and 48-hours post-treatment, respectively (Figure lb). Conversely, Dinutuximab beta was able to activate effector cells in an antibody-dependent-cellular cytotoxicity (ADCC) in vitro assay (Figures 1c and le). Taken together, these data show that Dinutuximab beta does not affect directly the viability and proliferation rate of Ewing's sarcoma cells, but it induces strong cytotoxicity by recruitment and activation of the immune system, in particular of the effector cells such as NK cells (Horta, Z.P., et al. Immunotherapy. 2016 Sep;8(9): 1097-117; Zeng, Y., et al. Mol Immunol. 2005 Jul;42(ll): 1311-9).

[0525] 1.3.2. Dinutuximab beta inhibits tumour growth and increases survival in vivo.

[0526] The anti-tumour activity of Dinutuximab beta was tested on a xenograft murine model of Ewing's sarcoma established by injecting subcutaneously TC-71 cells into nude athymic mice, as previously described (Amaral, A.T., et al. Clin Cancer Res. 2015 Mar 15;21(6): 1373-82; Belgiovine, C., et al. Br J Cancer. 2017 Aug 22;117(5):628-638). Nine days after tumour inoculation, mice were randomized into the following five treatment groups: Vehicle, Control IgG, Dinutuximab beta, Doxorubicin, and Combination, as described in Figure 2a. Animals in the Dinutuximab beta group received daily i.p. injections of Dinutuximab beta at 15mg / kg for five consecutive days, from day 0 to day 4 post-randomization; animals in the Doxorubicin group received the drug i.v. at 8mg / kg on day 0 and day 7, while in the Combination group, the animals received the two drugs following the same scheme as the single treatments. Two control groups were included: the first group received five consecutive daily i.p. injections of the vehicle, while the second control group received five consecutive daily i.p. injections of the control IgG isotype at 15mg / kg. In parallel, satellite groups for each treatment were sacrificed on day 4, four hours after the last dose of Dinutuximab beta, and on day 11, ninety-six hours after the second dose of Doxorubicin, for pharmacodynamic and pharmacokinetic studies.

[0527] Tumour growth curves obtained during the experiment are reported in Figure 2b. Compared with the vehicle group, Dinutuximab beta as single agent significantly inhibited tumour growth (p=0.0343 vs vehicle), but this effect was limited to the treatment period, then tumour regrowth occurred. The best mean tumour volume ratio between the treated and control groups (T / C) was 48% on day 13. The treatment with doxorubicin had a slightly better efficacy with a T / C of 36% on day 13 (p=0.0215 vs vehicle). The combination of Dinutuximab beta and doxorubicin improved the efficacy of the antibody alone, with a best T / C of 23% on day 13. This improvement is statistically significant when compared with Dinutuximab beta administered alone (p= 0.0067). Complete statistical analysis of the tumour growth curves is reported in table 8.

[0528] Table 8. Statical analysis of tumour growth curves.

[0529] Mixed-effects model (REML) P-value

[0530] CTRL vs Control IgG 0.241

[0531] CTRL vs Dinutuximab Beta 0.0343

[0532] CTRL vs Doxorubicin 0.0215

[0533] CTRL vs Combination 0.0027

[0534] Dinutuximab Beta vs Doxorubicin 0.3511

[0535] Dinutuximab Beta vs Combination 0.0067

[0536] Doxorubicin vs Combination 0.2394

[0537] The analysis of the survival curves (figure 2c) substantially confirmed the treatment efficacy, with increment of life span (ILS) of 45%, and 1 out of 9 mice being tumour- free at the end of the observation period, in the group of mice treated with Dinutuximab beta alone. The combined treatment further improved mice survival, resulting in an ILS of 95%. In the doxorubicin group, an ILS of 145% was recorded, probably related to the variability of the tumour regrowth after treatment. Complete statistical analysis of the tumour growth curves is reported in table 9.

[0538] Table 9. Statistical analysis of survival curves.

[0539] RMST (vs CNTRL) DIFF. ST.ERR. Z p-VALUE

[0540] Control IgG -1.67 2.67 -0.62 0.5327

[0541] Dinutuximab beta 10.67 3.76 2.84 0.0046

[0542] Doxorubicin 19.67 6.08 3.23 0.0012

[0543] Combination 19.78 3.57 5.55 0.0000

[0544] Dinutuximab beta vs Doxorubicin 9.00 6.78 1.33 0.1843

[0545] RMST (vs Combination) DIFF. ST.ERR. Z p VALUE

[0546] Dinutuximab beta 9.11 4.66 1.95 0.0506

[0547] Doxorubicin 0.11 6.67 0.02 0.9868

[0548] Figure 3 shows the growth curve of each individual tumour over time. All the tumours in the vehicle and control IgG groups increased in size over the treatment period. Conversely, the treatments caused varying degrees of partial tumour shrinkage. Specifically, on day 4, the last day of Dinutuximab beta treatment, both Dinutuximab beta and Doxorubicin as single treatments caused shrinkage of 3 out of 9 (30%) tumours, while the combined treatment caused shrinkage of 5 out 9 tumours (55%) (Figure 3b). Subsequently, tumours in the Doxorubicin and Combination groups regrew slower than those in the Dinutuximab beta group, likely due to the second dose of doxorubicin administered on day 7.

[0549] The treatment with Dinutuximab beta was well tolerated, with no body weight loss (BWL) or other drug-related clinical signs of distress (figure 3c). On the other hand, doxorubicin, both as monotherapy and in combination, caused significant BWL averaging around 10%, which is considered a sign of moderate suffering. In the doxorubicin group, one mouse was found dead for unknown reasons after a body weight loss of about 10%.

[0550] 1.3.3. Dinutuximab beta inhibits tumour cell proliferation and induces necrosis

[0551] Tumours of the satellite groups collected at the two time points (see Figure 2a) underwent histopathological evaluation after Hematoxylin & Eosin staining (Figure 4a). All samples showed signs of necrosis, with more necrotic tissue in control animals at T2 than in control animals at Tl, as expected due to massive tumour growth. The extension of necrotic areas was greater in all groups of treatment in comparison with controls at both timepoints. This increase was particularly evident in Dinutuximab beta and Combination groups at T2 (Figure 4b). Moreover, in untreated tumors, necrosis was primarily localized to the central region of the specimens at both time points, consistent with expected patterns of spontaneous necrosis. In contrast, tumors that received treatment, particularly in the Dinutuximab beta group, exhibited a diffuse distribution of necrosis throughout the entire tumor mass (Figure 4e and Figure 12). This finding confirms that the treatment with Dinutuximab effectively reaches the entire tumor mass.

[0552] The mitotic and apoptotic cell count revealed significant differences across the various treatment groups. Notably, there was an increase in the mitosis count in the group treated with Dinutuximab beta at Tl, although statistical significance was not reached (p=0.122, Kruskal-Wallis test). While this observation may seem contradictory to the overall tumour reduction, it can be explained by the possibility of tumour cells being arrested in the S and G2 / M phases, thus not actively proliferating but appearing morphologically similar to mitotic cells. By T2, there was a marked decrease in the mitotic cell count across all treatment groups, which correlates well with a strong reduction of tumour growth (Figure 4c). Apoptosis was increased in the group that received Doxorubicin or the combination, at both timepoints (Figure 4d). Taken together these data suggest that both Doxorubicin and Dinutuximab beta (and their combination) inhibit tumour cell proliferation. They also induce cell death, but through different mechanisms. Doxorubicin, either as monotherapy or in combination, induces apoptosis, as expected due to its mechanism of action (Kciuk, M., et al. Cells. 2023 Feb 19; 12(4): 659). Conversely, Dinutuximab beta exerts its cytotoxic effect mainly through necrosis, either as monotherapy or in combination. The two major therapeutic mechanisms described for Dinutuximab beta against GD2-positive cancer cells are antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which both trigger cell death through necrosis (Horta, Z.P., et al. Immunotherapy. 2016 Sep;8(9): 1097-117).

[0553] To gain a more comprehensive understanding of the mechanism of action of Dinutuximab beta, we performed RNA sequencing analysis on tumor biopsies from satellite groups treated with either vehicle or Dinutuximab beta, at both time points. We leveraged the difference in species origin to distinguish transcripts derived from the tumor (human) from those originating from host-infiltrating cells (murine). As expected, the majority of transcripts, ranging from 85% to 95%, were of human origin, while only 1.5% to 5.8% derived from the host (Figure 13). The analysis of the murine transcriptome identified four differently expressed transcripts (adjusted p Value < 0.05) at Tl (Figure 14a, Figure 15a-b), all upregulated following treatment with Dinutuximab beta. Gene ontology analyses (Figure 14b) revealed enrichment for biological processes involved in the response to cytokines responsible for triggering the acute phase of inflammation, as well as the immune response, including Interleukin-1 (IL-1) and Interferon-gamma. Parallelly, these genes are positively associated to chemotaxis and migration of immune cell populations, including lymphocytes, monocytes and granulocytes. These data confirm that Dinutuximab beta induces innate immune response through secretion of cytokines and recruitment of effector cells.

[0554] At T2, five protein-coding mRNA and one IncRNA up-regulated in the Dinutuximab beta group were identified (Figure 14c, Figure 15c-d). Here also, the gene ontology revealed enrichment for biological processes involved in response to cytokines and activation of the immune response, particularly of the adaptive immune system, as revealed by genes involved in antigen processing and presentation via MHC class II (Figure 14d). The analysis on human transcriptome identified two transcripts differentially expressed at T1 (Figure 15a-b). HERC2P8 is pseudogene with no know biological function, while IFITM3 is a gene that has been shown to modulate the tumor microenvironment by promoting T-cell infiltration, which is essential for antitumor immunity (Cai et al., 2021). In contrast, its deficiency disrupts antigen processing and presentation (Gomez- Herranz et al., 2019). Recently, IFITM3 has also been identified as a potent biomarker for predicting immunotherapy efficacy in some cancers and its overexpression has been shown to enhance antitumor immunity (Cui et al., 2025). At T2 thirty-seven (twelve upregulated and twenty-five downregulated following treatment with Dinutuximab beta) differentially expressed transcripts were identified (Figure 16a, Figure 17c-d). Of these, twenty-nine transcripts were protein-coding mRNA, showing enrichment in biological processes such as translational elongation and termination, cell aging and apoptosis (Figure 16b). Moreover, IFITM3 was also upregulated in Dinutuximab-treated tumors at T2. Interestingly, this upregulation is paralleled by enrichment of gene involved in antigen processing and presentation by murine effector cells infiltrating the tumor, suggesting a potential key role of this gene in the response to dinutuximab beta. These findings reinforce the hypothesis that the antitumor effect of Dinutuximab beta is mediated through the arrest of the cell cycle and the induction of cell death.

[0555] 1.3.4. A second cycle of treatment enhances the efficacy of combination therapy

[0556] As previously shown in Figure 2, Dinutuximab beta as a single agent induced a significant inhibition of tumour growth, which was, however, limited to the duration of the treatment. Following a single treatment with Dinutuximab beta, tumour re-growth was observed. Subsequently, the tumour began to grow again. Conversely, the treatment with doxorubicin had a slightly longer effect on tumour growth, which can be attributed to the second dose given on day 7. In fact, the growth curves of tumours treated with Dinutuximab beta or Doxorubicin begin to diverge after day 8.

[0557] As is typical for monoclonal antibodies, Dinutuximab beta has a long half-life, ranging from days to weeks (a half-life of 59 hours (4.5 days) in nude athymic mice (Gillies et al., 1993)). When administered intraperitoneally in mice at a single dose of 15mg / kg, it achieves a Cmax of 147.2 ug / ml after 6 hours and a Tl / 2 of 196 hours (8.2 days), as shown in Figure 5a. The concentration of Dinutuximab beta was measured in the plasma of tumour-bearing mice from the satellite groups, collected on day 4 and day 11 after treatment start (Figure 5b). It rapidly decreased from 933.1 ± 280.2 ug / ml 4-6 hours after the last administration (on day 4) to 115.2 ± 36.15 ug / ml on day 11, with a reduction of 87.7 % in 7 days (Tl / 2 3.9 days). This shorter half-life of the drug may be ascribed to its extensive binding to the tumour and subsequent elimination and / or metabolism. Whatever the mechanism, this rapid decrease of tumour exposition to Dinutuximab beta can explain the drug's short-term efficacy on tumour growth. To address this limitation, we designed a follow-up experiment in which the treatment with Dinutuximab beta was repeated a second time, 7 days after the start of the first cycle (day 7 to 11). Similarly, the combination therapy was administered in two cycles. Moreover, with the aim of enhancing the synergistic efficacy of the combination therapy, we included a group of animals that received a different treatment regime. In this group, the animals were initially treated with doxorubicin on day 0. Following a two-day interval, a 5-day cycle of Dinutuximab beta treatment started, spanning from day 2 to day 6. This regimen was then repeated for a second cycle, extending from day 9 to day 15. From now on, the two combination regimens will be referred to as "Simultaneous combination" and "Sequential combination", respectively. The detailed experimental scheme is shown in Figure 6a.

[0558] The tumour growth curves (Figure 6b) confirm the efficacy of Dinutuximab beta as single agent, and indicate that, when administered in two cycles, it is as effective as doxorubicin in inhibiting tumour growth. In fact, the two curves are superimposable, with the best T / C being 40% (p=0.0135 vs vehicle) and 36% (p=0.0189 vs vehicle; p=0.7417 vs Dinutuximab beta) on day 10 for the Dinutuximab beta and doxorubicin group, respectively. The effect of Dinutuximab beta was more evident during the first cycle of treatment, then regrowth occurred, and the second cycle of treatment did not further delay tumour growth i.e., only a minimal effect was observed.

[0559] In agreement with the first mouse experiment, the simultaneous combination of Dinutuximab beta and doxorubicin administered in two cycles improved the efficacy of Dinutuximab beta monotherapy, with the best T / C, on day 10, of 14% (p=0.0009 vs vehicle). This improvement was statistically significant compared to the use of Dinutuximab beta (p=0.0106) and Doxorubicin (p=0.0481) as monotherapies. The two distinct combination regimens showed comparable efficacy, with the best T / C being 18% (p=0.0018 vs vehicle, p=0.832 vs simultaneous combination) for the sequential combination group. Complete statistical analysis of the tumour growth curves is reported in table 10.

[0560] The analysis of the survival curves (figure 6c) confirmed the efficacy of Dinutuximab beta with an increment of life span (ILS) of 68% (p=0.0006 vs vehicle). In the doxorubicin group, a comparable ILS of 85.7% was recorded (p=0.0023 vs control). The superimposable activity of the two active agents was demonstrated by the absence of significant difference between their survival curves (p=0.725). The two combination treatment regimens further improved mice survival, resulting in an ILS of 146% (p=0.000025 vs vehicle) and 150% (p=0.0122 vs vehicle) for the simultaneous and sequential combination, respectively. However, the simultaneous combination was significantly better than either Dinutuximab beta or Doxorubicin as monotherapy, while the sequential combination was not. Complete statistical analysis of the tumour growth curves is reported in table 11.

[0561] Table 10. Statistical analysis of tumour growth curves of the two-cycle treatment.

[0562] P- Mixed-effects model (REML) value

[0563] CTRL vs Dinutuximab beta 0.0135

[0564] CTRL vs Doxorubicin 0.0189

[0565] CTRL vs Combination simultaneous 0.0009

[0566] CTRL vs Combination sequential 0.0018

[0567] Dinutuximab beta vs Doxorubicin 0.7417

[0568] Dinutuximab beta vs combination simultaneous 0.0106

[0569] Dinutuximab beta vs combination sequential 0.0248

[0570] Doxorubicin vs combination simultaneous 0.0481

[0571] Doxorubicin vs combination sequential 0.102

[0572] Combination simultaneous vs combination sequential 0.832 Table 11. Statistical analysis of survival curves of the two-cycle treatment.

[0573] RMST (vs CNTRL) DIFF. ST.ERR. Z p-VALUE

[0574] Dinutuximab beta 11.13 3.22 -3.45 0.0006

[0575] Doxorubicin 12.63 4.15 3.04 0.0023

[0576] Combination simultaneous 27.63 6.56 4.21 0.000025

[0577] Combination sequential 19.38 7.73 2.51 0.0122

[0578] Doxorubicin vs Dinutuximab beta 1.50 4.26 0.35 0.7250

[0579] RMST (vs Dinutuximab beta) DIFF. ST.ERR. Z p-VALUE

[0580] Combination simultaneous 16.5 6.64 2.49 0.0129

[0581] Combination sequential 8.25 8.00 1.03 0.3026

[0582] RMST (vs Doxorubicin) DIFF. ST.ERR. Z p-VALUE

[0583] Combination simultaneous 15 7.13 2.10 0.0354

[0584] Combination sequential 6.75 8.22 0.82 0.4115

[0585] Figure 7 shows the growth curve of each individual tumour over time. All the tumours in the control group increased in size over the treatment period. On day 5, after the end of the first cycle of treatment, Dinutuximab beta and doxorubicin monotherapies caused partial shrinkage of 1 (12.5%) and 2 (25%) out of 8 tumours, respectively. The "simultaneous combination" induced regression of 5 out of 8 tumours (62.5%) on day 5, which lasted for 4 out of the 5 regressed tumours (90%; 4 out of the total of 8, 50%) tumours on day 12, at the end of the second cycle of treatment (Figure 7b). Conversely, the "Sequential combination" was not as effective, inducing partial shrinkage in only 1 out of 6 (16.7%) tumours. Subsequently, tumours in the two combination groups re-grew slower than those in the monotherapy groups. Additionally, a single tumour in the simultaneous combination group was completely cured, with no signs of recurrence within the duration of the experiment. Two treatment cycles with Dinutuximab beta were well tolerated, with no BWL or other drug-related clinical signs of distress (figure 7c). As previously reported, doxorubicin caused significant BWL, averaging around 10%, when administered as a monotherapy and as a component in a combined therapy. In the "Sequential combination" group, one mouse was euthanised due to tumour ulceration, and 3 other mice reported a BWL greater than 10%, resulting in a total of 4 out of 7 animals (57%) experiencing health issues. Conversely, only 2 out of 8 mice (25%) in the "simultaneous combination" group reported a BWL greater than 10%. Taken together, these data suggest a better tolerability of the simultaneous combination regimen. 1.4. Discussion

[0586] In this study, we investigated the effects of Dinutuximab beta, a monoclonal antibody against the GD2 ganglioside, in combination with Doxorubicin, a widely used induction chemotherapeutic agent, on Ewing's sarcoma xenografts in mice. We found that Dinutuximab beta is as effective as doxorubicin in reducing tumour growth and increasing survival of the animals. Moreover, we found that the combination of chemo and immunotherapy further increased the therapeutic efficacy of the single therapies leading to a stronger inhibition of tumour growth.

[0587] Administered in a single cycle of five consecutive daily injections, Dinutuximab beta induced rapid tumour regression starting from the day after therapy initiation. This rapid therapeutic response could imply an initial activation of the complement system, which typically acts more quickly than effector cells like NK cells. However, once the treatment course concluded, tumours began to re-grow. A second cycle of treatment with Dinutuximab beta resulted in only a slight improvement in its effectiveness, showing almost no inhibition of tumour growth. On the other hand, when combined with chemotherapy, the second cycle significantly enhanced therapeutic efficacy, resulting in slower tumour progression and prolonged survival. Collectively, these findings indicate that combining Dinutuximab beta with Doxorubicin overcomes the loss of therapeutic efficacy of Dinutuximab beta when administered in multiple cycles, suggesting a synergistic effect that enhances antitumour efficacy. The histopathological analysis of tumours suggested that Dinutuximab beta and Doxorubicin induced different modes of cell death in tumour tissue. In fact, Dinutuximab beta caused predominantly necrosis, while Doxorubicin induced mainly apoptosis. The different mechanisms of action of the two agents may provide a rationale for their synergism, as they target different aspects of tumour biology.

[0588] The RNAseq analysis revealed the infiltration of effector cells of the innate immune response, likely driven by pro-inflammatory cytokines, in tumors treated with Dinutuximab beta. These findings confirm the proposed mechanism of action of Dinutuximab beta, which involves the recruitment of effector cells, mainly NK cells, to tumor sites. Here, effector cells exert their antitumor effect by inducing tumor cell death. The murine model used in the present study has some limitations. Although nude athymic mice retain a certain level of effector cells, including natural killer (NK) cells, the overall functionality of the innate immune response may be compromised compared to that of immunocompetent organisms. Consequently, the therapeutic efficacy of Dinutuximab beta observed in this model may underestimate its potential in patients with fully functional immune systems.

[0589] In this study, we examined the effectiveness of two different chemo-immunotherapy regimens by giving Doxorubicin and Dinutuximab beta either at the same time or one after the other. The findings suggest that administering them simultaneously results in a stronger tumour reduction and slightly reduced toxicity.

[0590] In clinical practice, following clinical trial outcomes and SIOPEN guidelines, Dinutuximab beta is administered to high-risk neuroblastoma patients as monotherapy after an intense consolidation phase of chemotherapy. Accordingly, Spasov and colleagues administered Dinutuximab beta after each cycle of consolidation chemotherapy in an uncontrolled study on 3 patients with either Ewing's sarcoma or Ewing's-like sarcoma (Spasov, N ., et al. 2022. J Pediatr Hematol Oncol. 2022 Aug I;44(6):e948-e953). Our findings indicate that administering Dinutuximab beta concurrently with Doxorubicin enhances tumour response. If confirmed by clinical studies, this evidence can change the future approach of chemo-immunotherapy treatment.

[0591] Overall, this study showed for the first time that Dinutuximab beta is effective against Ewing's sarcoma, especially in combination with Doxorubicin, a standard component of the induction chemotherapy regimen for Ewing's sarcoma. Further studies are needed to confirm the safety and efficacy of this combination in clinical trials.

[0592] In conclusion, the promising results of our preclinical experiments suggest a potential new therapeutic strategy for patients with Ewing's sarcoma. By combining immunotherapy with traditional chemotherapy, we can enhance treatment outcomes and improve patient survival rates. We eagerly anticipate the translation of these findings into clinical practice, paving the way for more effective cancer treatments.

[0593] Example 2

[0594] 2. Phase I trial of dinutuximab beta and vincristine / doxorubicin / cyclophosphamide and ifosfamide / etoposide in children, adolescents, and in GD2 positive Ewing sarcoma.

[0595] 2.1. Rationale

[0596] Immunotherapeutic approaches have emerged as an effective therapeutic approach in cancer. We will investigate the cell-surface ganglioside GD2 as a target since it is expressed on the cell surface of Ewing Sarcoma cells and clinical trials support the safety and efficacy of anti-GD2 antibodies in paediatric solid cancers.

[0597] Dinutuximab beta is a chimeric antibody that targets the disialoganglioside GD2. GD2 is expressed on a number of solid malignancies and was clinically investigated in paediatric patients with neuroblastoma. Expression in normal tissues is restricted to cerebellar neurons, skin melanocytes and peripheral pain fibers. Due to this expression pattern, anti-GD2 antibodies have been studied as targeted immunotherapy for neuroblastoma.

[0598] The administration of vincristine, doxorubicin, cyclophosphamide, ifosfamide and etoposide (VDC / IE) has been shown to be an effective systemic therapy and is therefore currently part of the standard treatment in Ewing Sarcoma. In the iEuroEwing trial, which opened very recently, in December 2022, alternating VC / IE is the new consolidation backbone in the standard- and high-risk group.

[0599] The rationale for this trial is to investigate the feasibility, toxicity, and biological activity of dinutuximab beta in combination with standard chemotherapy in EWS.

[0600] 2.2. Objective / hypothesis

[0601] Phase I single arm: Dose finding to determine the recommended phase II dose (R.PD2) of dinutuximab beta in combination with vincristine, doxorubicin & cyclophosphamide and ifosfamide & etoposide.

[0602] 2.3. Endpoints

[0603] 2.3.1. Primary endpoints Phase I: To determine a recommended dose of dinutuximab beta in combination with standard chemotherapy agents.

[0604] 2.3.2. Secondary Endpoints

[0605] Event Free survival (EFS)

[0606] Duration of Response (DOR)

[0607] 2.3.3. Exploratory Endpoints

[0608] • Assessment circulating tumour DNA baseline and at each course

[0609] • Gene signatures in whole genome data

[0610] • Test of tumour microenvironment

[0611] • Test of immune response in peripheral blood (FACS)

[0612] Biobanking of liquid biopsies, stool, and tumour material for ancillary studies

[0613] 2.4. Investigational medicinal products (IMP):

[0614] Dinutuximab beta, Vincristine, Cyclophosphamide, Ifosfamide, Etoposide, Doxorubicin

[0615] 2.5. Dinutuximab beta dosing

[0616] Phase I

[0617] Recommended dose: 10mg / m2 / d 10-day continuous infusion

[0618] The starting dose

[0619] Level 1: 50% of the recommended dose; 5mg / m2 / d 10-day continuous infusion

[0620] Level 2: 75% of the recommended dose; 7.5mg / m2 / d 10-day continuous infusion

[0621] Level 3: 100% of the recommended dose; 10mg / m2 / d 10-day continuous infusion

[0622] 2.6. Inclusion criteria phase I

[0623] 1. Histologically confirmed, newly diagnosed EWS (m / f / d) with evidence of EWS translocation by fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), or next-generation sequencing (NGS) assay

[0624] 2. High risk stratification (e.g. metastatic disease)

[0625] 3. Centrally confirmed GD2-positive tumor (biopsy of original and / or recurrent tumor)

[0626] 4. Age >12 months 5. Start of firstline treatment according to iEuroEwing standard (Cycle 1-4: VDC - IE - VDC - IE)

[0627] 6. Karnofsky (> 16 years) or Lansky (<16 years) Performance Score >70% or ECOG 2

[0628] 7. Adequate bone marrow function as evidenced by meeting all the following requirements: a. White blood cell count > 2000 / pl b. ANC >500 cells / pL without the use of hematopoietic growth factors within the last 2 days c. Platelet count 75,000 cells / pL without the use of platelet transfusion within the last 2 days d. Hemoglobin >9 g / dL without the use of red blood cell transfusion within the last 2 days

[0629] 8. Adequate hepatic function as evidenced by meeting all the following requirements: a. Serum total bilirubin <1.5 x upper limit of normal (ULN) b. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 5 x ULN

[0630] 9. Adequate cardiac function:

[0631] Left ventricular ejection fraction (LVEF) > 40%, shortening fraction (SF) > 28%

[0632] 10. No known active HIV, HBV, or HCV infection

[0633] 11. Female patients of childbearing potential must present with a negative serum pregnancy test and agree to employ adequate birth control measures for the duration of the study and until 3 months after the end of treatment. Female patients who are lactating must agree to stop breast-feeding from the start of study treatment until 1 month after the end of treatment.

[0634] 12. Patient or their legal representative is willing and able to comply with the requirements of the study protocol

[0635] 2.7. Exclusion criteria phase I:

[0636] 1. Patients with hypersensitivity against at least 1 component of the investigational medicinal product or against mouse proteins

[0637] 2. Previous treatment with an anti-GD2 antibody

[0638] 3. Significant illnesses and / or any of the following :

[0639] • significant psychiatric disabilities or uncontrolled seizure disorders

[0640] • active uncontrolled peptic ulcer disease

[0641] • clinically significant neurologic deficit or objective peripheral neuropathy

[0642] • clinically significant, symptomatic pleural effusions • Active and uncontrolled CNS metastases (indicated by clinical symptoms, cerebral edema, corticosteroid and / or anticonvulsant requirement, or progressive disease); for controlled CNS metastases, patient should have been off corticosteroids for at least 28 days without overt evidence of significant neurological deficits prior to enrollment

[0643] 4. Significant cardiac conduction abnormalities, including known familial prolonged QT syndrome, or screening QTc >480 msec

[0644] 5. Symptoms of congestive heart failure or left ventricular ejection fraction <40%

[0645] 6. Active, uncontrolled infection or an unexplained fever >38.5°C which in the Investigator's opinion might compromise the patient's participation in the study or affect the study outcome

[0646] 7. Chronic Grade >2 diarrhea

[0647] 8. Diagnosis of any malignancy other than the disease under study

[0648] 9. Female patient who is pregnant or lactating at the time of enrolment

[0649] 10. Any other medical or social condition deemed by the Investigator to be likely to interfere with a patient's ability to cooperate and participate in the study or interfere with the interpretation of the results.

[0650] 2.8. Definitions

[0651] The evaluation of the tumour will be performed according to the revised RECIST guideline (version 1.1).

[0652] Complete response: Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm

[0653] Partial response (PR): At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.

[0654] Stable disease: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.

[0655] Progressive disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression). Example 3

[0656] 3. Multicentre, International, randomized, parallel, open label Phase II trial evaluating the efficacy and safety of the combination of dinutuximab beta with standard chemotherapy in Ewing Sarcoma relapsed patients

[0657] 3.1. Primary objectives

[0658] To assess the efficacy of Dinutuximab beta (Db) combined with standard chemotherapy regimen on event-free survival (EFS) compared to a standard chemotherapy regimen alone in patients with relapsed GD2+ Ewing Sarcoma.

[0659] 3.2. Secondary objectives

[0660] 1. To evaluate efficacy of Dinutuximab beta in addition to standard chemotherapy on overall survival (OS).

[0661] 2. To evaluate efficacy of Dinutuximab beta in addition to standard chemotherapy on overall response rate (ORR) by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST vl.l).

[0662] 3. To evaluate efficacy of Dinutuximab beta in addition to standard chemotherapy on duration of response (DOR).

[0663] 4. To assess safety of Dinutuximab beta in combination with standard chemotherapy

[0664] 5. To assess the efficacy of Dinutuximab beta based on patients' reported outcome (PRO) questionnaire and on health-related quality of life (HRQoL) questionnaire.

[0665] 6. To assess anti-drug antibodies (ADA) and Dinutuximab beta pharmacokinetic (PK).

[0666] 7. To assess complement dependent cytotoxicity (CDC).

[0667] 8. To assess Immuno-phenotyping.

[0668] 9. To validate a method for GD2 expression detection in solid / liquid biopsies.

[0669] 3.3. Primary endpoint

[0670] Event-free survival (EFS) defined as the time from randomization to the occurrence of the first event among local or distant disease progression, relapse, occurrence of a second malignancy, discontinuation of the treatment for toxicity, or death from any cause. 3.4. Secondary endpoints

[0671] 1. Overall Survival (OS) defined as the time from randomization to death from any cause.

[0672] 2. Overall Response Rate (ORR) assessed by RECIST 1.1 defined as the proportion of patients who have a partial response (PR) or complete response (CR) to therapy.

[0673] 3. Duration of Response (DOR) defined as time (in days) from the first occurrence of a response (CR or PR, which was subsequently confirmed) until the date of progression or death prior to progression.

[0674] 4. Incidence of AEs, SAEs, laboratory, and ECG findings. Changes from baseline in laboratory values and in vital signs.

[0675] 5. Changes in HRQoL and PRO from baseline to the end of treatment.

[0676] 6. Measure Db serum concentration immediately pre-Db infusion (Cthrough) and immediately post Db infusion (C max) .

[0677] 7. Measure ADA to Db immediately pre-Db infusion.

[0678] 8. Measure CDC immediately pre and post Db infusion.

[0679] 9. Measure immunophenotyping response immediately pre Db and immediately post Db infusion. Where available the following tests should be performed: a. T-cells (CD3+) b. Activated NK cells (CD56 DIM / + / CD16+) c. Regulatory T-cells (CD4+ / CD25+ / CD127-) d. Activated granulocytes & monocytes (CD64+) e. Absolute NK-cells (CD56+ / CD3-)

[0680] 10. GD2 expression may be assessed.

[0681] 3.5. Trial design

[0682] This is a multicentre, international, randomized, open label, phase II trial evaluating the efficacy and safety of Dinutuximab beta in combination with standard chemotherapy versus standard chemotherapy alone in patients with relapsed GD2+ Ewing sarcoma with a 2-year follow up. The intervention assignment will be by 1 : 1 randomisation after screening.

[0683] An interim analysis will be performed when 50% of the total number of events planned for the primary efficacy analysis is reached, to check on superiority-based drug efficacy based on EFS.

[0684] 3.6. Inclusion criteria 1. The patient or legal representative is willing and able to give written informed consent for participation in the study before performance of any study-specific screening procedures.

[0685] 2. Histologically confirmed, Ewing Sarcoma (m / f / d) or Ewing-like sarcoma (i.e. translocation-positive small blue round cell sarcoma other than Rhabdomyosarcoma) of bone and I or soft tissue with evidence of EWS translocation by fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), or next-generation sequencing (NGS) assay.

[0686] 3. Documented disease relapse (such as at least 6 months from end of prior therapy) and no more than 1 prior line of therapy.

[0687] 4. Centrally confirmed GD2-positive tumour (biopsy of original and / or residual tumour or liquid biopsy in peripheral blood and biopsy at the time of relapse).

[0688] 5. Availability of tumour tissue for central GD2-detection.

[0689] 6. Age > 2 years.

[0690] 7. Life expectancy > 3 months.

[0691] 8. Patients candidate for treatment for relapsed disease.

[0692] 9. Measurable disease present according to RECIST 1.1.

[0693] 10. Wash-out phase with a minimum of 14 days after the last the dose of the last chemotherapy regimen in relapsed patients.

[0694] 11. Prior radiotherapy is allowed if:

[0695] • > 2 weeks have elapsed for local palliative external beam radiotherapy.

[0696] • > 6 months must have elapsed if systemic radiotherapy, external craniospinal irradiation or > 50% pelvic radiotherapy; and

[0697] • > 6 weeks must have elapsed for other substantial bone marrow radiotherapy before the first dose.

[0698] 12. Participants who have received brain radiotherapy must have completed whole brain radiotherapy and / or gamma knife surgery at least 4 weeks prior to enrollment.

[0699] 13. Lansky (< 16 years) Performance Score (PS) >70% or Eastern Cooperative Oncology Group (ECOG) PS of (>16 years) 0-2.

[0700] 14. Symptomatic CNS metastases must have been treated and remain stable for at least 4 weeks prior to the first dose of the study drug and off corticosteroids.

[0701] 15. Adequate bone marrow function as evidenced by meeting all the following requirements: a. White blood cell count > 2000 / pL b. ANC >1000 cells / pL (G-CSF allowed) c. Platelet count 75,000 cells / |_il_ without the use of platelet transfusion within the last 2 days d. Haemoglobin >9 g / dL without the use of red blood cell transfusion within the last 2 days

[0702] 16. Adequate hepatic function as evidenced by meeting all the following requirements: a. Serum total bilirubin <1.5 x upper limit of normal (ULN) b. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 5 x ULN.

[0703] 17. Adequate cardiac function: confirmed by echocardiography with a left ventricular ejection fraction (LVEF) of > 50%.

[0704] 18. Adequate renal function: creatinine clearance or glomerular filtration rate (GFR) > 60 mL / min / 1.73 m2.

[0705] 19. No known active HIV, HBV, or HCV infection.

[0706] 20. No severe neurological impairment, particularly no motor or sensory deficits, except for neurological deficits caused by Ewing sarcoma.

[0707] 21. Childbearing potential and contraception: a. Female patients of childbearing potential must present with a negative serum pregnancy test and agree to employ adequate birth control measures for the duration of the study and until 3 months after the end of treatment. Female patients who are lactating must agree to stop breast-feeding from the start of study treatment until 1 month after the end of treatment. b. Males who are sexually active must agree to use a condom with spermicidal foam / gel / film / cream / suppository during the study and for at least 90 days after the last study drug administration agree to not donate sperm during the study and for at least 90 days after the last study drug administration no plan to father a child during the study and for at least 90 days after the last study drug administration.

[0708] 22. Patient or their legal representative is willing and able to comply with the requirements of the study protocol.

[0709] 3.7. Exclusion criteria

[0710] 1. Relapse within 6 months from a prior systemic therapy.

[0711] 2. Current participation in another clinical trial.

[0712] 3. No more than 1 prior line of therapy. 4. Patients with hypersensitivity against at least 1 component of the investigational medicinal product or against mouse proteins .

[0713] 5. Prior Unresolved, > Grade 1 toxicity related to prior anti-tumour therapy prior to the study, according to the CTCAE version 5.0.

[0714] 6. Significant illnesses and / or any of the following : a. Significant psychiatric disabilities or uncontrolled seizure disorders b. Active uncontrolled peptic ulcer disease c. Clinically significant neurologic deficit or objective peripheral neuropathy d. Clinically significant, symptomatic fluid in a third space e. Active and uncontrolled CNS metastases (indicated by clinical symptoms, cerebral oedema, corticosteroid and / or anticonvulsant requirement, or progressive disease); for controlled CNS metastases, patient should have been off corticosteroids for at least 28 days without overt evidence of significant neurological deficits prior to enrolment. f. Significant cardiac conduction abnormalities, including known familial prolonged QT syndrome, or screening QTc >480 msec. g. Symptoms of congestive heart failure or left ventricular ejection fraction <40%. h. Active, uncontrolled infection or an unexplained fever >38.5°C which in the Investigator's opinion might compromise the patient's participation in the study or affect the study outcome. i. Chronic Grade >2 diarrhoea. j. Diagnosis of any malignancy other than the disease under study, except for basal cell carcinoma of the skin or carcinoma in situ that has been completely resected.

[0715] 7. Female patient who is pregnant or lactating at the time of enrolment.

[0716] 8. Any other medical or social condition deemed by the Investigator to be likely to interfere with a patient's ability to cooperate and participate in the study or interfere with the interpretation of the results.

[0717] 9. Concomitant treatment with: a. Intravenous immunoglobulins b. Vaccinations c. Other systemic anticancer treatment not described in this protocol d. Corticosteroids.

[0718] 3.8. Trial population Male and female patients > 2 years of age with histological and biomarker proof of relapsed Ewing's sarcoma, with GD2 expression (central), meeting all inclusion criteria and presenting no exclusion criteria at screening.

[0719] 3.9. Test product

[0720] Dinutuximab beta (mouse-human chimeric monoclonal IgGl antibody), which may be administered at a dose of 10mg / m2 / day for 10 days per cycle.

[0721] 3.10. Control

[0722] Chemotherapy according to local standard of care (e.g., TOPOCYCLO: Cyclophosphamide & Topotecan; or TEMIRI: Temozolomide & Irinotecan; or high dose Ifosfamide).

[0723] 3.11. Primary hypothesis

[0724] There will be 2 arms in the trial: i. interventional arm with standard chemotherapy (proposed to be TEMIRI or TOPOCYCLO chemotherapy, according to standard of care) plus dinutuximab beta; ii. control arm with standard chemotherapy alone (proposed to be TEMIRI or TOPOCYCLO chemotherapy, according to standard of care).

[0725] The primary hypothesis to be tested is that Dinutuximab beta plus standard chemotherapy (CT) leads to a better Event Free Survival (EFS) compared with standard CT alone in relapsed Ewing Sarcoma patients.

[0726] EFS is defined as the time from randomization to the occurrence of the first event among local or distant disease progression, relapse, occurrence of a second malignancy, discontinuation of the treatment for toxicity, or death from any cause. Example 4

[0727] 4. Pre-clinical evaluation of the efficacy of Dinutuximab beta in addition to the standard chemotherapy backbone against Ewing Sarcoma

[0728] References to Dinutuximab in the figures associated with the following example should be taken to mean "Dinutuximab beta" as described in the sub-section 4.2 entitled "Materials and methods".

[0729] 4.1. Introduction

[0730] Ewing sarcoma is an aggressive cancer of bone which forms preferentially in femur and pelvis, and sometimes in soft tissues surrounding bones. It is rare, with an incidence of 1.5 cases per million in teenagers. Around 25% of patients present at diagnosis with metastasis and have an overall 5-year survival rate lower than 30%. It has been reported that around 80% of metastatic and / or recurrent Ewing's sarcomas express GD2. Dinutuximab beta (Qarziba, Recordati) is a human-mouse chimeric monoclonal IgGl against GD2 approved for the treatment of pediatric patients with high-risk neuroblastoma. Preclinical studies in preclinical models of Ewing sarcoma are needed to assess efficacy and tolerability of Qarziba in addition to the standard of care for this tumor histotype.

[0731] The aim of this study is to evaluate the efficacy of Qarziba in addition to standard-of- care chemotherapy for Ewing sarcoma, which includes Doxorubicin (D), Cyclophosphamide (C), Vincristine (V), Ifosfamide (I), and Etoposide (E).

[0732] 4.2. Materials and methods

[0733] 4.2.1. Drugs

[0734] Qarziba (Dinutuximab Beta, Q) was provided by Recordati S.p.a. , D, C, V, I and E were purchased by MedChemExpress, and prepared accordingly to the relative instructions supplied together with the compounds.

[0735] 4.2.2. Cell line

[0736] TC71 cell line carrying the EWS / FLU type I translocation were maintained in Iscove's modified Dulbecco's medium supplemented with 10% Fetal Bovine serum, 2 mM glutamine, and 1% Penicillin-Streptomycin. 4.2.3. Mice

[0737] Female Athymic nude mice, 7-8 weeks old, were obtained from INOTIV. They were maintained under Specific Pathogen Free conditions with constant temperature and humidity, according to the institutional guidelines.

[0738] 4.2.4. Tumor model

[0739] To obtain the tumors, 200 pl of cell suspension containing 5 x 106TC71 cells were injected subcutaneously into the right flanks of the mice. The growing tumor masses were measured with the aid of a Vernier caliper, and the tumor weight (1mm3=lmg) was calculated by the formula: length x (width)2 / 2.

[0740] When tumor weight reached about 100 mg, mice were randomized in the following experimental groups (at least 8 mice for each group):

[0741] • Vehicles

[0742] • Qarziba (Dinutuximab beta)

[0743] • Chemotherapy dose low (Chemo low)

[0744] • Chemotherapy dose high (Chemo high)

[0745] • Chemo low + Dinutuximab beta (Combo low)

[0746] • Chemo high + Dinutuximab beta (Combo high)

[0747] The therapeutic regiment was administered according to the scheme in Figure 18.

[0748] The administration route as well as the doses of each drug are summarized in table 12. The dose of each drug was decided based on the results of previous studies.

[0749] Table 12

[0750] * Intraperitoneal injection (i.p.); Intravenous therapy (i.v.) Tumor bearing mice were monitored daily for body weight loss (BWL) and clinical signs of distress, and tumor growth was assessed, by Vernier caliper, at least twice a week. Mice were euthanized when tumors reached a volume of > 1500 mm3, in case of tumor ulceration or BWL >20%. The antitumor activity was expressed as T / C%, where T and C were the mean tumor weight of treated and control groups, respectively.

[0751] 4.3. Results

[0752] The anti-tumour activity of Dinutuximab beta was tested on a xenograft murine model of Ewing's sarcoma established by injecting subcutaneously TC-71 cells into nude athymic mice, as previously described (Amaral et al., 2015; Belgiovine et al., 2017). Nine days after tumour inoculation, mice were randomized into the following six treatment groups: Vehicle, Dinutuximab beta, Chemo low dose, Chemo high dose, Combination low dose and combination high dose as described in Figure 18. Animals in the Dinutuximab beta group received two cycles of daily i.p. injections of Dinutuximab beta at 15mg / kg for five consecutive days, from day 0 to day 4 (cycle 1) and from day 7 to day 11 (cycle 2) post-randomization; animals in groups Chemo low and Chemo high received V, D and C on day 0 and I and E on day 7 at the doses indicated in Table 12. In the two Combination groups, the animals received the chemotherapeutics and Dinutuximab beta following the same scheme as the single treatments. The Vehicle group received two cycles (day 0-4 and day 7-11) of five consecutive daily i.p. injections of the vehicle.

[0753] Tumour growth curves obtained during the experiment are reported in Figure 19a. Compared with the vehicle group, Dinutuximab beta as single agent slightly inhibited tumour growth (p=0.57 vs vehicle), with a best T / C of 65% on day 11. The treatments with both Chemo low and high significantly inhibit tumor growth, with the highest dose being more effective. The best T / C was 32% (p=0.0571 vs vehicle) for Chemo low and 25% (p=0.0239 vs vehicle) for Chemo high on day 11. The combination of Dinutuximab beta and chemotherapy further improved the anti-tumor efficacy of chemo- and immune-therapy alone, with the combination with the highest dose of chemotherapy being the most effective. The best T / C was 16% (p=0.0037 vs vehicle) and 6% (p=0.0002 vs vehicle) for Chemo low and Chemo high respectively. This improvement is statistically significant when compared with either Dinutuximab beta or chemotherapy administered alone. Complete statistical analysis of the tumour growth curves is reported in Table 13. Table 13. Statistical analysis of tumour growth curves.

[0754] The analysis of the survival curves (figure 19b) substantially confirmed the treatment efficacy, with significantly increment of life span (ILS) of 82% (p=0.0057 vs vehicle), and 1 out of 9 mice being tumour-free at the end of the observation period, in the group of mice treated with Combo high.

[0755] Figure 19c illustrates the change in size of each individual tumor at day 11, marking the end of the treatment period. As expected for this type of aggressive disease, all tumors in the vehicle group increased in size during treatment. Monotherapies, either Dinutuximab or chemotherapy, resulted in no or only limited partial tumor shrinkage. Conversely, the combination therapy induced tumor regression in 5 out of 9 tumors (56%) at the low dose and in 8 out of 9 tumors (89%) at the high dose (Figure 19c). All the treatments well tolerated, with no major body weight loss (BWL) or other drug- related clinical signs of distress (Figure 19d).

[0756] 4.4. Conclusions In conclusion, the combination of Dinutuximab beta with the standard-of-care chemotherapy backbone VDC / IE demonstrated significant therapeutic benefit in Ewing sarcoma preclinical model, achieving substantial tumour regression in most treated animals, particularly at the higher dose. Monotherapies showed minimal efficacy, highlighting the advantage of the combination approach. Importantly, all treatment regimens were well tolerated, with no notable body weight loss or overt signs of distress observed. These findings corroborate the evidence that combining Dinutuximab beta with chemotherapy represents a promising strategy for improving outcomes in patients affected by Ewing sarcoma.

[0757] Example 5

[0758] 5. Multicentre, International, randomized, open label Phase III trial evaluating the efficacy of dinutuximab beta in combination with irinotecan and temozolomide in Ewing Sarcoma relapsed patients

[0759] Example 5 describes a proposed Phase III clinical trial to evaluate the efficacy of dinutuximab beta in combination with irinotecan and temozolomide in Ewing Sarcoma relapsed patients. The trial design is based on the protocol outlined in Example 3, with the following modifications indicated below:

[0760] 5.1. Primary endpoint

[0761] Overall survival (OS) defined as the time from randomization to death from any cause.

[0762] 5.2. Secondary endpoint

[0763] Progression-free survival (PFS) is defined as the time from random assignment in a clinical trial to disease progression or death from any cause. PFS as assessed per BICR without inclusion of a second malignancy.

[0764] 5.3. Trial design

[0765] A futility analysis may be conducted prior to any efficacy analysis and may be performed ahead of the primary PFS analysis. Sample size re-estimation may be performed during or as a consequence of this analysis. 5.4. Trial population

[0766] The patient cohort may include up to 188 patients.

[0767] 5.5. Inclusion criteria • Disease relapse includes patients experiencing an initial relapse, a second relapse or any subsequent relapse.

[0768] • Age > 4 years.

[0769] • Patient candidate may also have progressive disease. 5.6. Trial protocol

[0770] Patients will be randomized 1: 1 to receive dinutuximab beta 10 mg / m2 / day as a 10- day continuous infusion with irinotecan 50 mg / m2IV over 60 minutes and temozolomide 100 mg / m2orally (PO) on days 1-5 in 21-day cycles vs TEMIRI alone. There will be no crossover planned during the study. Treatment will continue on either arm for up to 12 cycles. Patients will be stratified by time to relapse (<2 years vs ~2 years) and site of recurrence (local vs lung vs others). Tumor assessments will be performed every 2 cycles. Patients will be followed for 2 years.

[0771] The dosing strategy is outlined in Figure 10.

Claims

1. CLAIMS1. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy of:(i) administration to the subject of an anti-GD2 antibody; and(ii) administration to the subject of at least one treatment dose of induction chemotherapy with one or more chemotherapeutic agent(s).

2. The method according to claim 1, wherein the subject has relapsed Ewing sarcoma; optionally, wherein the subject has relapsed 6 or more months after:(i) a remission diagnosis;(ii) the completion of the previous therapy regimen; and / or(iii) commencement of maintenance therapy.

3. The method according to claim 1, wherein the subject has high-risk metastatic Ewing sarcoma, particularly newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma, optionally wherein the cancer has metastasised from the bone or soft tissue to at least one other site within the body.

4. The method according to any one of claims 1-3, wherein the subject has not previously received treatment with an anti-GD2 antibody for the treatment of Ewing sarcoma; optionally wherein the subject has not previously been treated with an anti- GD2 antibody for any purpose.

5. The method according to any of the preceding claims, wherein the anti-GD2 antibody is administered at a dose of from about 1 mg / m2 / d to about 20 mg / m2 / d; optionally wherein the anti-GD2 antibody is administered at a dose of from about 4 mg / m2 / d to about 10 mg / m2 / d, such as at a dose from about 5 mg / m2 / d to about 10 mg / m2 / d or at a dose from about 4 mg / m2 / d to about 8 mg / m2 / d.

6. The method according to any of the preceding claims, wherein the anti-GD2 antibody is administered at a dose of about 7.5 mg / m2 / d or at a dose of about 5 mg / m2 / d.

7. The method according to any of the preceding claims, wherein the anti-GD2 antibody is a chimeric anti-GD2 antibody; optionally, wherein the anti-GD2 antibody is Dinutuximab or Dinutuximab beta; further optionally, wherein the antibody has the heavy chain sequence SEQ ID NO: 19 and the light chain sequence SEQ ID NO: 18.

8. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination induction chemotherapy according to any one of Claims 1-7, followed by a combination consolidation chemotherapy of:(i) administration to the subject of an anti-GD2 antibody; and(ii) administration to the subject of at least one treatment dose of consolidation chemotherapy with one or more chemotherapeutic agent(s).

9. A method of treating relapsed GD2-positive Ewing sarcoma, particularly newly diagnosed relapsed GD2-positive Ewing sarcoma, in a subject in need thereof, the method comprising a combination chemotherapy of:(i) administration to the subject of an anti-GD2 antibody; and(ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or any combinations thereof.

10. The method according to claim 9, wherein the Ewing sarcoma has relapsed after 6 or more months after:(a) a remission diagnosis;(b) the completion of the previous therapy regimen; and / or(c) commencement of maintenance therapy.

11. A method of treating GD2-positive Ewing sarcoma in a subject in need thereof, the method comprising a combination chemotherapy of:(i) administration to the subject of an anti-GD2 antibody, wherein the dose of anti-GD2 antibody is selected from 4-8 mg / m2 / d; and(ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or any combinations thereof.

12. The method according to claim 11, wherein the anti-GD2 antibody is administered at a dose of from about 5 mg / m2 / d to about 7.5 mg / m2 / d, optionally at a dose of 5 mg / m2 / d or at a dose of 7.5 mg / m2 / d.

13. A method of treating high-risk metastatic GD2-positive Ewing sarcoma, particularly newly diagnosed high-risk metastatic GD2-positive Ewing sarcoma, in a subjectwherein at least one metastatic tumoral site is distally located from the lung, the method comprising a combination chemotherapy of:(i) administration to the subject of an anti-GD2 antibody; and(ii) administration to the subject of at least one treatment dose of chemotherapy with one or more chemotherapeutic agent(s), selected from the group consisting of induction chemotherapy, consolidation chemotherapy, maintenance chemotherapy, non-standard chemotherapy or any combinations thereof.

14. The method according to any one of the previous claims, wherein prior to the combination chemotherapy, the method comprises the step of determining or confirming that the subject has GD2-positive Ewing sarcoma, optionally wherein the subject has at least one confirmed GD2 positive tumour, biopsy or cell sample; further optionally wherein the step of determining or confirming that the subject has GD2- positive Ewing sarcoma comprises one or more of the following : immunostaining, flowcytometry, RT-PCR, or ELISA.

15. The method according to any previous claim, wherein the subject does not have refractory Ewing sarcoma.111

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