EA043975B1 - DRUGS AND TREATMENT METHODS FOR GD2-POSITIVE CANCER - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to preparations and methods for treating GD2-positive cancer by administering to a patient a preparation containing a chimeric or humanized anti-GD2 antibody without the patient also receiving interleukin-2 (IL-2). In particular, the invention relates to preparations and methods for treating GD2-positive cancer by administering to a patient a preparation containing a chimeric or humanized anti-GD2 antibody, wherein the patient does not simultaneously receive interleukin-2 (IL-2), and in some embodiments, also does not receive granulocyte colony-stimulating factor -macrophages (GM-CSF) and/or one or more other cytokines. The invention further provides drugs and methods for treating GD2-positive cancer in a patient, wherein the drug containing an anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days without concomitant administration of IL-2. The invention also provides preparations and methods for treating GD2-positive cancer in a patient by administering to the patient a preparation containing an anti-GD2 antibody without simultaneous administration of IL-2, wherein one or more periods of antibody administration are preceded, followed and/or followed by one or more periods of retinoid administration. The invention also relates to drugs and methods for treating GD2-positive cancer by administering a drug containing an anti-GD2 antibody as a continuous infusion to a patient over 24 hours a day, wherein the patient does not simultaneously receive interleukin-2 (IL-2), either alone or The multiple antibody administration periods may be preceded, followed, and/or followed by one or more retinoid administration periods.

Prior Art

After brain cancer, neuroblastoma is the most common form of solid cancer in children under five years of age. In high-risk neuroblastoma, more than half of patients receiving standard treatment relapse and eventually die from the disease. 90% of cases occur between ages zero and six years. Worldwide incidence in industrialized countries is approximately 2000 cases per year.

In oncology, monoclonal antibodies against certain antigens are increasingly used. A completely different mode of action compared to cytotoxic therapy makes them a valuable asset, as shown by their first representatives such as trastuzumab, cetuximab, bevacizumab, rituximab, etc. Disialoganglioside GD2 is a glycosphingolipid that is expressed mainly on the cell surface. In normal tissues, GD2 expression is rare and is mainly limited to the central nervous system (CNS), peripheral nerves, and melanocytes. In cancer cells, GD2 is expressed uniformly in neuroblastomas and most melanomas and to varying degrees in bone and soft tissue sarcomas, small cell lung cancer, renal cell carcinoma, and brain tumors (Navid et al., Curr. Cancer Drug Targets, 2010, 10: 200-209). Due to its relatively selective expression in tumors coupled with its presence on the cell surface, GD2 represents a promising target for antibody-based cancer immunotherapy.

Accordingly, several anti-GD2 antibodies are in preclinical or clinical studies in neuroblastoma, melanoma, and other GD2-related cancers.

APN311 is a chimeric anti-GD2 monoclonal antibody ch14.18 produced recombinantly in Chinese hamster ovary (CHO) cells, which are the standard mammalian cell line for producing commercially available antibodies. In a phase I clinical trial, patients with relapsed/refractory neuroblastoma went into remission with this antibody as a single agent. A phase III trial involving APN311 was initiated by the International Society of Pediatric Oncology and European Neuroblastoma (SIOPEN) in 2006 and is currently investigating its effect on relapse-free survival and overall survival when APN311 is used with isotretinoin, i.e. cis-retinoic acid (cis-RA), with or without subcutaneous IL-2. In a comparable study in the United States, treatment with a 4-drug package of a related antibody produced in murine hybridoma cells SP2/0, together with IV interleukin-2 (IL-2 or IL2), granulocyte-macrophage colony-stimulating factor (GM- CSF) and isotretinoin, there was an interesting improvement in survival in children with neuroblastoma in complete remission after initial therapy and without evidence of disease.

APN301 is an immunocytokine preparation containing a humanized antibody against GD2 (hu14.18) and IL-2 as a fusion protein. The antibody moiety specifically binds to the GD2 antigen, which is highly expressed in neuroblastoma and several other cancers. IL-2 is a cytokine that recruits several types of immune effector cells. In neuroblastoma patients, APN301 targets GD2-positive tumor cells via an antibody component. The IL-2 fusion then stimulates the patient's immune system against the tumor by activating NK cells and T cells, while the Fc region of the antibody serves to trigger tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The immunocytokine was active in a phase II clinical trial in children with relapsed/refractory neuroblastoma (Shusterman et al., JCO, 2010, 28(33):4969-75), and was also tested in a phase I/II trial in advanced cancer.

- 1 043975 melanoma, showing activation of the immune system.

Other anti-GD2 antibodies that are in the process of research or development are, for example, the monoclonal antibody 3F8 (murine in phase II, also humanized in phase I) and 8B6 (specific to O-acetylated GD2, preclinical). In addition, anti-idiotypic antibodies, such as 4B5, 1A7 and A1G4, were studied as potential anti-tumor vaccines, but their development was apparently abandoned. WO 2008/049643 also describes anti-idiotypic antibodies that mimic GD2 epitopes, i.e. These are GD2 mimeotopes.

Another variant of the 14.18 anti-GD2 antibody is hu14.18K322A, as described in WO 2005/070967, which contains a point mutation in the Fc region to attenuate CDC but retains ADCC, for example, when expressed in a cell line suitable for enhancing ADCC, such as YB2/0. It is believed that a decrease in CDC results in a decrease in pain associated with antibody use.

The antitumor activity of antibodies usually occurs through either complement-dependent cytotoxicity (CDC or complement fixation) or antibody-dependent cellular cytotoxicity (ADCC). These two activities are known in the art as effector functions and are mediated by antibodies, particularly those of the IgG class. All IgG subclasses except IgG4 (IgG1, IgG2, IgG3) mediate ADCC and complement fixation to some extent, but the strongest for both activities are IgG1 and IgG3. ADCC is thought to occur when Fc receptors on natural killer (NK) cells and/or other Fc receptor-bearing immune cells (effector cells) bind to the Fc region of antibodies bound to an antigen on the cell surface. Binding to Fc receptors gives a signal to effector cells to destroy target cells. CDC is thought to occur through several mechanisms; one mechanism is triggered by the binding of an antibody to an antigen on the cell surface. Once the antigen-antibody complex is formed, the C1q molecule is believed to bind to the antigen-antibody complex. C1q is then itself cleaved, triggering a cascade of enzymatic activation and cleavage of other complement proteins, which then bind to the surface of target cells and promote their death, for example, through cell lysis and/or ingestion by macrophages.

Antibody-dependent cellular cytotoxicity (ADCC) is believed to play an important role in immunotherapy. Unfortunately, ADCC is often suppressed in cancer patients. Cytokines are thought to enhance ADCC by directly activating immune cells or by upregulating tumor-associated antigens (TAAs) on tumor cells. For example, aldezleukin (IL-2) causes the activation of natural killer (NK) cells, the formation of lymphokine-activated killer (LAK) cells, and enhances ADCC. Aldezleukin (IL-2) was effective in inducing measurable antitumor responses in patients with renal cell carcinoma and melanoma. In addition, GM-CSF has been shown in vitro and in vivo to enhance antitumor immunity through direct activation of monocytes, macrophages, dendritic cells and antibody-dependent cellular cytotoxicity (ADCC) and indirect activation of T cells through TNF, interferon and interleukin-1 (LL -2). GM-CSF is thought to enhance cell functions critical for immune activation against tumor cells, alone or with other cytokines or monoclonal antibodies.

In summary, ongoing clinical trials investigating antibodies against GD2, particularly ch14.18, combine the use of antibodies with the use of cytokines (and retinoids), especially IL-2 and/or GMCSF. Accordingly, the prior art teaches that patients with GD2-positive cancer benefit from administration of cytokines, particularly in combination with the use of anti-CD2 antibodies.

On the contrary, one of the key aspects of the invention is that such patients can be treated with anti-GD2 antibodies without IL-2, in particular without the use of any cytokines.

Treatment with one or more cytokines in combination with an antibody may have serious side effects such as fever, allergic reactions, hypotension, capillary leak syndrome, etc., which may even lead to death. Concomitant treatment with cytokines even potentiates side effects from the use of antibodies, such as pain, since there is synergy in the adverse effects of both drugs. However, with the drugs and methods of the present invention, any cytokines can be completely eliminated. Thus, the present invention results in a significant reduction in the adverse effects of antibody treatment.

The essence of the invention

In a first aspect, the present invention relates to preparations containing an anti-GD2 antibody for use in the treatment of GD2-positive cancer in patients, wherein the preparation containing a chimeric or humanized anti-GD2 antibody is administered to the patient without simultaneous administration of IL-2, thereby treating GD2- positive cancer.

The invention further relates to drugs and methods for treating GD2-positive cancer in a patient, wherein the drug containing a chimeric or humanized anti-GD2 antibody is administered to the patient as a continuous infusion over 24 hours per day without simultaneous administration of IL-2. The present invention also provides drugs and methods for treating GD2-positive cancer in a patient, wherein the drug containing a chimeric or humanized anti-GD2 antibody is administered to the patient without concomitant administration of IL-2, with one or more periods of administration of the anti-GD2 antibody

- 2 043975 precedes, accompanies and/or follows one or more periods of retinoid administration. In some embodiments, the patient does not receive GM-CSF and/or one or more other cytokines, especially not within the same treatment cycle.

The invention is further defined by specific embodiments and claims. All embodiments of the invention, as described below, apply to all aspects of the invention equally and all these aspects can be combined, for example, the first with the second or third, the first with the second and third, the second with the third, giving a preparation for the method from the combined elements of these aspects; or giving combined methods.

Brief description of the figures

In fig. Figure 1 shows Kaplan-Meier curves for relapse-free survival (EFS, Fig. 1A) and overall survival (OS, Fig. 1B) based on data (as a percentage over time) from 328 patients with neuroblastoma who received APN311 and isotretinoin, but without IL- 2 (light gray), and patients treated with APN311 and isotretinoin with IL-2 (dark gray).

In fig. Figure 2 compares EFS data (percentage over time) from the 128 patients from Example 2 who had a complete response (CR) at the start of treatment (Fig. 2A, right panel) with EFS data (percentage over time) from Yu et al. 2010, New England Journal of Medicine, 363:1324-1334 (Figure 2A, left panel, also includes fully susceptible data). In fig. Figure 2B shows an overlay of diagrams for comparison.

In fig. Figure 3 compares the OS data (percentage over time) from the 128 patients from Example 2 who had a complete response (CR) at the start of treatment (Fig. 3A, right panel) with the OS data (percentage over time) from Yu et al., 2010 (Figure 3A, left diagram). In fig. 3B shows an overlay of diagrams for comparison.

In fig. 4 and 5 present summary tables of toxicity (Fig. 4: for all levels of toxicity and separately only grades 3 and 4; Fig. 5: for all levels of toxicity for each treatment cycle), expressed as a percentage of the total number of patients examined who received treatment according to the appropriate regimen with and without IL-2.

In fig. Figures 6 and 7 present the corresponding diagrams for toxicity (Fig. 6: for all levels of toxicity; Fig. 7: only grades 3 and 4) noted in all treatment cycles, as a percentage of the total number of patients examined who were treated according to the corresponding regimen with IL -2 and without it.

In fig. 8 and 9 show charts for all levels of toxicity for each treatment cycle as a percentage of the total number of patients examined who were treated according to the corresponding regimen with IL-2 (Fig. 9) and without IL-2 (Fig. 8).

In fig. 10-15 are charts of the toxicities observed in the respective treatment cycles (Fig. 10: cycle 1; Fig. 11: cycle 2; Fig. 12: cycle 3; Fig. 13: cycle 4; Fig. 14: cycle 5; Fig. 15: cycle 6), as a percentage of the total number of patients examined who were treated according to the appropriate regimen with and without IL-2.

In fig. 16 shows the results of a complement dependent cytotoxicity (CDC) assay (large black circles, FIG. 16A) and a whole blood assay (large black circles, WBT, FIG. 16B) from blood samples from a patient with neuroblastoma during treatment with anti-GD2 antibody but without IL. -2 with the intake of cis-retinoic acid. The white circles outlined in black are samples from patient samples treated with an anti-idiotypic antibody to distinguish from any possible nonspecific lysis (i.e., lysis of target cells that is not mediated by antibodies). White stripes circled around the perimeter in black indicate periods of antibody administration.

In fig. 17 shows the results of a complement dependent cytotoxicity (CDC) assay (large black circles, FIG. 17A) and a whole blood assay (large black circles, WBT, FIG. 17B) from blood samples from a patient with neuroblastoma during treatment with anti-GD2 antibody with a regular dose of IL. -2 and taking cis-retinoic acid. Black-circled white circles are samples from patient samples treated with an anti-idiotypic antibody to distinguish from any possible nonspecific lysis (i.e., lysis of target cells that is not mediated by antibodies). White stripes circled around the perimeter in black indicate periods of antibody administration, white triangles circled around the perimeter in black indicate periods of taking IL-2, and black asterisks indicate periods of taking cis-retinoic acid.

Detailed Description of the Invention

Contrary to expectations, treatment with one or more cytokines in combination with an anti-GD2 antibody and a retinoid did not provide any clinical benefit over treatment with an anti-GD2 antibody and a retinoid but without IL-2, or without IL-2 and without GM-CSF, or even without any cytokine. In addition, a treatment regimen without IL-2 or other cytokines in combination with a continuous infusion of an anti-GD2 antibody drug has been shown to be very beneficial, particularly in reducing side effects, especially pain.

In one aspect, the invention provides a preparation containing an anti-GD2 antibody (also referred to as an antibody preparation) for use in the treatment of GD2-positive cancer in patients, wherein the preparation containing an anti-GD2 antibody is administered to the patient without concomitant administration of IL-2,

- 3 043975 this involves treating a GD2-positive cancer in a patient.

In another aspect, the invention provides a method of treating GD2-positive cancer in a patient, comprising administering to the patient a preparation comprising an anti-GD2 antibody without concomitant administration of IL-2, thereby treating the GD2-positive cancer in the patient.

In another aspect, the invention relates to the use of an anti-GD2 antibody in the preparation of a medicament for the methods provided herein.

In some embodiments, the anti-GD2 antibody is administered to the patient as a continuous infusion over 24 hours per day. In some embodiments, the anti-GD2 antibody is administered to a patient as a continuous infusion over one or more days. In some embodiments, the patient receives the retinoid one or more times before, during, and/or after administration of the anti-GD2 antibody. In some embodiments, the anti-GD2 antibody is a chimeric or humanized anti-GD2 antibody.

The term patient in the present invention means a human patient suffering from GD2-positive cancer. The term treating or being treated in the present invention means that a drug or treatment is administered to a patient in need thereof.

The term “treating a GD2-positive cancer in a patient” means that a therapeutic effect is achieved in that patient, as further defined below.

The terms "co-treated" or "co-administered" as used herein mean that one treatment (e.g., with an anti-GD2 antibody and/or a preparation containing an anti-GD2 antibody, referred to as antibody administration) is preceded, accompanied, and/or followed by one or more other treatments. (such as, for example, taking one or more analgesics and/or one or more other drugs or agents), particularly within the same treatment cycle and/or within the same total treatment time (for example, when in which the anti-GD2 antibody is administered). The concomitant treatment period may not coincide with another treatment period (eg, the antibody administration period), either partially or completely. Accordingly, a period of concomitant treatment (eg, analgesic administration) may precede, accompany, and/or follow a period of other treatment (eg, a period of antibody administration). In one embodiment, the concomitant treatment periods fall within the same treatment cycle.

Accordingly, the terms not treated simultaneously, without simultaneous administration or not administered simultaneously in the present invention mean that one treatment is not preceded, accompanied and/or followed by one or more other treatments, respectively. In one embodiment, the above terms mean that the patient does not receive a given drug or agent (ie, the drug or agent is not administered to a given patient) within the same treatment cycle and/or within the same total treatment time. Accordingly, the period of such non-concomitant therapy (eg, cytokine administration) should not overlap with another treatment period (eg, the period of antibody administration), either partially or completely. In one embodiment, the period of the non-concomitant treatment should not precede, accompany and/or follow the period of the other treatment (eg, the period of administration of the antibody). In one embodiment, the periods of non-concomitant treatment do not fall within the same treatment cycle. However, a patient who is not receiving a concurrent drug or agent (eg, one or more cytokines) may have received the drug or agent (eg, one or more cytokines) in previous treatment cycles and/or throughout the previous treatment.

The term cytokines as used herein means those proteins, peptides or glycoproteins that act as hormonal regulators or signaling molecules in nanomolar or picomolar concentrations and are involved in cellular signaling. In one embodiment, the one or more cytokines are selected from immunomodulatory agents, such as interleukins and/or interferons. In one embodiment, one or more cytokines is selected from the group consisting of IL-2, GM-CSF, granulocyte colony-stimulating factor (G-CSF), IL-12 and/or IL-15. In one embodiment, one or more cytokines are not fused to an antibody, in particular not to an anti-GD2 antibody.

The term reduced dose or low dose in the present invention refers to those doses of the corresponding drug that are significantly lower, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% (or any interval between these doses) is lower than the usual dose of the same drug when administered under the same or similar conditions, i.e. in the same or similar groups of patients with the same or similar treatment. A usual dose may be a dose that has been used frequently in the past and/or is primarily used in the same or similar conditions, i.e. in the same or similar groups of patients with the same or similar treatment. The dose may be reduced by reducing the daily dose and/or by reducing the frequency and/or duration of administration. For example, to reduce the dose by 50%, the corresponding drug can be administered at the same frequency or duration as usual, but at only half the usual daily dose, or the corresponding drug can be administered at the usual daily dose, but, for example, only every other day if he usually

- 4 043975 was entered every day. In another example, 50% of the usual daily dose may be administered every other day instead of the usual daily dose, which will result in the dose being reduced to 75% less than the usual dose. Accordingly, one skilled in the art will readily be able to determine suitable dosages and administration schedules in accordance with the invention.

A period of treatment with a particular drug or agent in the present invention means a period of time in which that particular drug or agent is administered to a patient within one treatment cycle, for example, a period of time of consecutive days of treatment. For example, if a cytokine-containing drug is typically administered for 5 consecutive days, followed by one or more days without taking the cytokine drug, then the cytokine-containing drug treatment period is 5 days. In another example, if a drug containing an anti-GD2 antibody is administered continuously for 24 hours for 10 consecutive days, followed by one or more days without administration of the drug containing an anti-GD2 antibody, then the treatment period with the drug containing an anti-GD2 antibody is is 10 days. In another example, if isotretinoin is administered twice daily for 14 days, followed by one or more drug-free days, then the isotretinoin treatment period is 14 days. Any such treatment periods may be repeated, overlap in whole or in part with other periods of treatment with other drugs or agents, and/or may be preceded and/or followed by periods without treatment. For example, a treatment cycle could be two 5-day IL-2 treatment periods, with the second overlapping with a 10-day (or 14, 15, or 21-day) ch14.18 (APN311) treatment period and then 14 -day period of treatment with isotretinoin.

The terms combined or combination as used herein in relation to treatment periods means that two or more treatment periods with the same and/or different drugs or agents are contained within a single treatment cycle. These two or more periods of treatment with different drugs or agents may partially or completely overlap or may not overlap. Any such treatment periods may be combined with (or separated by) one or more periods without the same and/or different drugs or agents.

The term treatment cycle in the present invention means a course of one or more treatments or treatment periods that are repeated on a regular basis, optionally with periods of rest (no treatment) between them. For example, treatment that is given for one week followed by three weeks of rest constitutes one treatment cycle. In one embodiment, one treatment cycle includes one period of treatment with a drug containing an anti-GD2 antibody. A treatment cycle comprising one period of treatment with a drug containing an anti-GD2 antibody may additionally include one or more periods of treatment with one or more other drugs or agents (excluding cytokine treatment), such as, for example, retinoids and/or analgesics. Any such periods of treatment with one or more drugs or agents within the same treatment cycle may overlap completely and/or partially. A treatment cycle may also include one or more periods of time without any treatment. The rest periods, for example, may be at least 1 day, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13 or 14 days or more. On the other hand, periods of rest, for example, may be no more than 8, 7, 6, 5, 4, 3, 2, 1 week or less. Each treatment in a treatment cycle may preferably be a treatment in accordance with the first, second, third, or some combination aspect as defined in the summary. The treatment regimen according to the invention may include 1 or more treatment cycles, in particular two or more, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more treatment cycles.

The terms total treatment time or entire treatment period in the present invention mean a continuous treatment period including one or more successive treatment cycles. The treatment cycle may be repeated, either in the same form or in a modified form, for example, with a different dose or schedule, or with one or more different and/or additional treatments (for example, with one or more other analgesics). The total treatment time may be at least 1 or 2 or several cycles, for example up to 10 or 20 or even more treatment cycles. In one embodiment, the total treatment time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles or more. As described above, treatment cycles may include non-treatment periods (intervals in which no agent, ie, antibody, cytokine, or other drug, is administered to the patient). Thus, in the present invention, the total treatment time may also include these non-treatment intervals within a treatment cycle and/or between treatment cycles. In one embodiment, a treatment cycle may immediately follow a previous treatment cycle, i.e. without a break between treatment cycles. However, the end of a treatment cycle may include a period of time of treatment before the start of the next treatment cycle. Examples of total treatment time are, for example, at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30 months or more.

If any range is given here, any such range shall include any space between the range limits (ie, the lower and upper limits of the range). For example, if a range is given, such as 1 to 5 days, then this should include 1, 2, 3, 4 and 5 days. That

- 5 043975 the same applies to any other ranges, including without limitation other time periods (for example, infusion time in hours), any dose ranges (for example, per 1 m ² body surface area, per 1 kg of body weight, per day , per 1 cycle of treatment, etc.), infusion rates, concentrations, percentages, coefficients, ratios and numbers.

Anti-GD2 antibody or antibody preparation can be administered to patients in need. In one embodiment, the patient is a GD2-positive cancer patient. GD2-positive cancer is a type of cancer in which GD2 is expressed on tumor cells. GD2-positive cancers include, for example, neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung cancer, desmoplastic small round cell fibroma, osteosarcoma, rhabdomyosarcoma and/or other soft tissue sarcoma. In one embodiment, the patient has been diagnosed with neuroblastoma, particularly high-risk neuroblastoma. In one embodiment, the patient was diagnosed with stage 4 neuroblastoma (according to the International Neuroblastoma Staging System (INSS)). In one embodiment, the patient was diagnosed with minimal residual disease. In one embodiment, the patient is diagnosed as fully receptive, i.e. the patient is fully responsive to treatment. In another embodiment, the patient has been diagnosed with a relapsed or refractory disease. In one embodiment, the patient suffers from primary refractory or recurrent high-risk neuroblastoma or high-risk minimal residual neuroblastoma disease. The patient may have previously undergone or may be simultaneously undergoing one or more other therapies, such as, for example, surgery, chemotherapy, radiation, myeloablative therapy, metaiodobenzylguanidine (mIBG) scintigraphy, vaccine therapy, stem cell transplant, and/or treatment with retinoids (eg, isotretinoin ).

In one embodiment, the patient is not enrolled in a phase I, II, or III clinical trial. In another embodiment, the patient is not enrolled in any clinical trial. In particular, the patient is not enrolled in any clinical trial in any country in the world. Accordingly, the patient does not participate in any systematic research and/or officially supported (for example, by any competent national or regional health authority) testing in the field of medical research and drug development that generates data on the safety and effectiveness of any drug or treatment . In one embodiment, the patient does not participate in any systematic research and/or officially supported (eg, any competent national or regional health authority) testing of any medical interventions (including diagnostics, devices, etc.). Previous clinical trials described in the prior art with anti-GD2 antibody without concomitant administration of IL-2 and/or any other cytokines were conducted to investigate the overall effects, adverse effects and doses of the antibody as a basis for further study. However, the prior art clearly teaches that patients should be treated with an anti-GD2 antibody in combination with at least one cytokine, especially IL-2. Accordingly, any currently used anti-GD2 antibody treatment regimens include at least one cytokine, particularly IL2, and also in combination with GM-CSF (eg, see Yu et al., cited above).

In one embodiment, the anti-GD2 antibody of the invention specifically binds to the GD2 antigen. GD2 is a disialoganglioside that is expressed on tumors of neuroectodermal origin. The antibody may be selected from the group of recombinant or artificial antibodies, including single chain antibodies, mammalian antibodies, human or humanized antibodies. It may contain or be selected from the constant and/or variable regions of the antibody, in particular from Fc, Fc-like, Fv, Fab, F(ab) ₂ , Fab', F(ab') ₂ , scFv, scFc, VHH. However, any such antibody fragments should thereby be able to mediate natural (or in vivo) effector functions. Preferably, the antibody contains an antibody light and a heavy chain. An antibody may contain one or two antigen-binding regions that may bind the same or different antigens, for example GD2, which may bind specifically. Antibodies according to the invention can be directed, for example, produced during immunization, against the antigens listed above. The anti-GD2 antibody may be a humanized or chimeric anti-GD2 antibody, such as a humanized or chimeric antibody 14.18, 3F8 or 8B6, or a murine antibody of the same specificity, or an antigen binding fragment of any of these, which mediates natural effector functions. In one embodiment, the antibody is not the 14G2a antibody. An anti-GD2 antibody may contain one or more amino acid modifications, such as a modified Fc region. In one embodiment, the anti-GD2 antibody is hu14.18K322A. In another embodiment, the anti-GD2 antibody is chimeric antibody 14.18. In one embodiment, the anti-GD2 antibody is encoded by the nucleic acid sequence of SEQ ID NO: 1 and/or 2 and/or the amino acid sequence of SEQ ID NO: 3 and/or 4, or fragments or homologues thereof. In one embodiment, the anti-GD2 antibody is encoded by the amino acid sequence of SEQ ID NO: 3 and/or 4 or fragments or homologues thereof.

The term fragments or homologues thereof means those fragments or homologues of the corresponding sequence that have the same or similar qualitative activity to the native one. In one

- 6 043975 embodiment fragments or homologs have at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% of the original qualitative activity. In one embodiment, the fragments or homologues are at least 80, 85, 90, 95, 96, 97, 98 or 99% identical in sequence.

In one embodiment, the anti-GD2 antibody has a light chain nucleotide sequence of SEQ ID NO: 1 (see also Example 1) and a heavy chain nucleotide sequence of SEQ ID NO: 2 (see also Example 1). In one embodiment, the anti-GD2 antibody has a light chain amino acid sequence of SEQ ID NO: 3 (see also Example 1) and a heavy chain amino acid sequence of SEQ ID NO: 4 (see also Example 1). The relative molecular weight of an antibody consisting of two light and two heavy chains may be approximately 150,000 daltons. In one embodiment, the anti-GD2 antibody preparation is APN311. In one embodiment, the anti-GD2 antibody is dinutuximab. The anti-GD2 antibody can be expressed in CHO cells, in SP2/0 cells or in other suitable cell lines, such as HEK-293, MRC-5, Vero, PerC6 or NS0. In one embodiment, the anti-GD2 antibody is antibody 14.18. In one embodiment, the anti-GD2 antibody is a chimeric antibody 14.18 expressed in SP2/0 cells (ch14.18/SP2/0). In another embodiment, the anti-GD2 antibody is a chimeric antibody 14.18 expressed in CHO cells (ch14.18/CHO). In one embodiment, the anti-GD2 antibody may be a fragment or homologue of the antibody encoded by SEQ ID NO: 1, 2, 3 and/or 4, with the same or similar qualitative activity.

In some embodiments, the anti-GD2 antibody is not fused to any other molecule. In some embodiments, the anti-GD2 antibody is not an immunocytokine. In some embodiments, the anti-GD2 antibody-containing preparation does not contain immunocytokines. In some embodiments, the patient does not simultaneously receive immunocytokines, particularly not within the same treatment cycle and/or throughout the entire treatment period including administration of the antibody.

The anti-GD2 antibody formulation may further contain salts and water for injection (WFI), and optionally amino acids, in particular basic amino acids such as histidine, arginine and/or lysine. In one embodiment, the anti-GD2 antibody preparation may further contain a buffer, for example, phosphate-buffered saline containing the indicated salts and WFI. The anti-GD2 antibody formulation may further contain stabilizing agents, preservatives and other carriers or excipients. In one embodiment, the anti-GD2 antibody-containing preparation contains an anti-GD2 antibody (eg, ch14.18) and further contains sucrose, polysorbate 20, histidine and hydrochloric acid. In one embodiment, the antibody is ch14.18/CHO and the antibody formulation is APN311 (reformulated), the formulation containing 4.5 mg/ml antibody, 50 mg/ml sucrose, 0.1 mg/ml polysorbate 20 and 3.1 mg/ml histidine. The anti-GD2 antibody preparation can be lyophilized. The reconstituted solution may have a pH of 6±0.5. In one embodiment, the anti-GD2 antibody preparation further contains sucrose, L-arginine, citric acid monohydrate, polysorbate 20 and hydrochloric acid. In one embodiment, the preparation contains 4 mg/ml anti-GD2 antibody, 20 mg/ml sucrose, 13.9 mg/ml L-arginine, 2 mg/ml polysorbate 20 and 2.1 mg/ml citric acid monohydrate. In one embodiment, the preparation is lyophilized and can be reconstituted in 4 ml of 0.9% sodium chloride, and the resulting solution has a pH of 5.5 (the pH can be adjusted with hydrochloric acid (HCl)). In one embodiment, the anti-GD2 antibody preparation does not contain stabilizing agents, preservatives or other excipients. The anti-GD2 antibody preparation may be added to an infusion bag, such as an infusion bag containing normal saline, i.e. saline NaCl (0.9%), optionally with human serum albumin (HAS, also called human albumin). In one example, the infusion bag contains normal saline and 0.25-5% human serum albumin, or any concentration between these concentrations. In one example, the final volume of the infusion bag contains 250 ml of 0.9% NaCl and 5 ml of 20% human albumin, or 100 ml of 0.9% NaCl and 5 ml of 20% human albumin, or 50 ml of 0.9% NaCl and 2 ml 20% human albumin.

An anti-GD2 antibody or a preparation containing an anti-GD2 antibody can be administered in daily antibody doses of 1 to 30 mg/ ^m2 , 1 to 35 mg/ ^m2 , 1 to 50 mg/m2 ^, or 1 to 60 mg/m2. ^m2 , for example 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 12, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 25, 30, 32, 35, 40, 45, 50 or 60 mg/ ^m2 or anywhere in between. In one embodiment, the anti-GD2 antibody or anti-GD2 antibody-containing preparation is administered in daily antibody doses of 7, 7.5, 8, 9, 10, 12, 13, 14, 15, 16, 17, 17.5, 18, 19 , 20, 25, 30, 32 or 35 mg/ ^m2 or at any interval between these doses. For example, a daily dose of 10 mg/m ² means that the patient receives 10 mg of anti-GD2 antibody per 1 m ² of body surface per day. In the present invention, dose (eg, given in mg or μg) refers to the dose of the active ingredient, i.e. the amount of active ingredient in the drug. For example, the dose may refer to the amount of an anti-GD2 antibody in a preparation containing an anti-GD2 antibody, or a cytokine in a preparation containing a cytokine, or morphine or other analgesic in a preparation containing morphine or another analgesic, etc. As stated in the example above, a daily dose of 10 mg/m ² means that the patient receives 10 mg of anti-GD2 antibody (which optionally contains

- 7 043975 in a certain volume of a preparation containing an antibody against GD2) per 1 m ² of body surface per day. In the present invention, the dose given per 1 m ² means per 1 m ² body surface area (BSA) of the patient. In the present invention, the dose given per 1 kg means per 1 kg of the patient's body weight.

In some embodiments, the anti-GD2 antibody-containing preparation is administered at daily doses of 1 to 15, 1 to 20, 1 to 25, 1 to 30, or 1 to 35 mg/m ² or any interval between these daily doses. doses. In some embodiments, the anti-GD2 antibody drug is administered in daily doses of less than 50.0, 30, or 25 mg/m ² . In some embodiments, the anti-GD2 antibody preparation is administered in daily doses of up to 7, 10, 15, 17.5 or 20 mg/m ² . In one embodiment, the antibody preparation is administered at a dose of 15, 17.5, 20 or 25 mg/m ² /day for 4 days. In one embodiment, the antibody preparation is administered at a dose of 50 mg/m ² /day for 4 days. The anti-GD2 antibody can be administered at a dose of 10, 20, 25, 50, 60, 65, 68, 70, 75, 80, 100, 120, 150, 200, 210, 250 or 300 mg/ ^m2 /cycle or at any interval between these doses. The total dose per patient per treatment cycle may be defined as the target total dose per patient.

In some embodiments, the anti-GD2 antibody-containing drug is administered as an intravenous infusion over 5 hours or more per day, e.g., 5.75 hours or more per day, 8 hours or more per day, 10 hours or more per day, or up to 20 hours a day or up to 24 hours a day, or any time in between, for example, for 4 or 5 days or more. In other embodiments, the anti-GD2 antibody-containing drug is administered as a continuous intravenous infusion over 24 hours per day. In some embodiments, the anti-GD2 antibody is administered as a continuous intravenous infusion over 24 hours per day until a predetermined total dose has been administered to the patient. In some embodiments, the anti-GD2 antibody is administered as a continuous intravenous infusion over 24 hours per day for one or more days, such as 4, 5, 10, 14, 15, or up to 21 or more days.

In some embodiments, a drug containing an anti-GD2 antibody is administered throughout the treatment period until a certain therapeutic effect is achieved. In some embodiments, the therapeutic effect may be an enhancement of the immune response to the tumor, as determined, for example, by an increase in immune system biomarkers (eg, blood parameters such as lymphocyte count and/or NK cell count; and/or cytokines). In some embodiments, the therapeutic effect may be a reduction in tumor markers (eg, catecholamines). In some embodiments, the therapeutic effect may be determined by methods such as metaiodobenzylguanidine (mIBG) scintigraphy, magnetic resonance imaging (MRI) or X-ray computed tomography (CT) and/or bone marrow histology (from aspirate or trephine biopsy) and/or tests CDC and/or WBTs.

In some embodiments, a therapeutic effect may be defined as a stable state (i.e., no further increase in lesions, tumor tissue, and/or size), a partial response (i.e., a decrease in lesions, tumor tissue, and/or size), and/or a complete response (i.e. complete remission of all lesions and tumor tissue).

Total reaction (CR) can be further defined as follows:

complete disappearance of all measurable and assessable foci of disease;

absence of new lesions;

absence of symptoms of the disease; and/or absence of measurable signs of disease, including, for example, normalization of markers and/or other abnormal laboratory values.

In some embodiments, all measurable, assessable and non-assessable lesions and sites should be assessed using the same methodology as initially. Partial reaction (CR) can be further defined as follows:

Applicable only to patients with at least one measurable lesion;

a decrease of more than or 50% from the original sum of the products of the perpendicular diameters of all measurable lesions;

absence of progression of this disease;

absence of new lesions.

In some embodiments, all measurable, assessable and non-assessable lesions and sites should be assessed using the same methodology as initially.

The anti-GD2 antibody preparation can be administered as described in PCT/EP2012/061618 or PCT/EP2012/064970. For example, a drug containing an anti-GD2 antibody can be administered as a continuous intravenous infusion over 24 hours per day. Accordingly, in one aspect, the invention provides preparations containing an anti-GD2 antibody for use in the treatment of GD2-positive cancer in patients, wherein the preparation containing the anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days and over one, two or more cycles of treatment, without simultaneous administration of IL-2. In another aspect, the invention relates to preparations containing an anti-GD2 antibody for use in the treatment of GD2-positive cancer in patients, wherein the preparation containing an anti-GD2 antibody is administered to a patient.

- 8 043975 entu as a continuous infusion without simultaneous administration of IL-2, and wherein the anti-GD2 antibody is not a 14G2a antibody or wherein the anti-GD2 antibody is a chimeric or humanized anti-GD2 antibody.

The anti-GD2 antibody formulation may be administered for 4, 5, 10, 14, 15 or 21 consecutive days or any time in between. A drug containing anti-GD2 antibody can also be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21 or more days in a row. In some embodiments, the anti-GD2 antibody-containing drug is administered throughout the entire treatment cycle, for example, for 35 days. In some embodiments, the anti-GD2 antibody-containing drug is administered as a continuous intravenous infusion for the duration of treatment, for example, over 5 treatment cycles of 35 days each, i.e. for a total of 180 days. The daily dose of antibody can be reduced accordingly so that a given dose of antibody is administered to the patient. In one embodiment, the predetermined dose of antibody is 100 mg/m ² /cycle. In one embodiment, the total treatment time is 5 cycles. Accordingly, in this example, the dose of antibody over the total treatment time is 500 mg/m ² . In one embodiment, this total antibody dose of 500 mg/m ² is administered over a total treatment period of 180 days, i.e. 2.77 mg/ ^m2 /day. The drug containing the anti-GD2 antibody can be administered as a continuous intravenous infusion over a period of 24 hours per day. An osmotic minipump can be used for this continuous infusion. In one embodiment, the anti-GD2 antibody-containing preparation is administered as a continuous intravenous infusion over 24 hours per day for 4, 5, 10, 14, 15 or 21 consecutive days or any time therebetween, at the daily dosages indicated above (e.g. 7, 10, 15, 17.5, 20 or 25 mg/ ^m2 /day), e.g. 15, 17.5, 20 or 25 mg/ ^m2 /day for 4 days, 20 mg/ ^m2 /day for 5 days, 10 mg/ ^m2 /day for 10 days, 15 mg/ ^m2 /day for 10 days, 7 mg/ ^m2 /day for 14 days, 15 mg/ ^m2 /day for 14 days, 10 mg/m ² /day for 15 days, 7 mg/m ² /day for 21 days, or 10 mg/m ² /day for 21 days or any interval between these doses. In some embodiments, the anti-GD2 antibody preparation is administered as a non-continuous intravenous infusion over 5 days at a daily dose of 40 mg/m ² . In some embodiments, the anti-GD2 antibody-containing drug is not administered as a continuous intravenous infusion over 5 days, i.e. not as a 120-hour infusion. In some embodiments, the anti-GD2 antibody-containing drug is administered as a continuous intravenous infusion over 5 days. In some embodiments, the anti-GD2 antibody-containing drug is administered as a continuous intravenous infusion over 6 days or more.

In one embodiment, the anti-GD2 antibody formulation is APN311 and is administered at a dose of 10 mg/m ² /day for 10 days. In one embodiment, the anti-GD2 antibody formulation is APN311 and is administered at a dose of 10 mg/m ² /day for 10 consecutive days over 1, 2, 3, 4, 5 or 6 or more treatment cycles.

In accordance with the present invention, the patient does not simultaneously receive IL-2. In some embodiments, the patient receives neither IL-2 nor GM-CSF simultaneously. In some embodiments, the patient does not receive any cytokines at the same time. Accordingly, the term the patient is not simultaneously receiving IL-2, GM-CSF and/or other cytokines includes the appropriate treatment regimens described above, i.e. without IL-2, without IL-2 and without GM-CSF and/or without any cytokines. In one embodiment, the patient does not simultaneously receive IL-2, GM-CSF, and/or other cytokines within the same treatment cycle. . In one embodiment, the patient does not simultaneously receive IL-2, GM-CSF, and/or other cytokines within the same total treatment time. In some embodiments, the patient may have received IL-2, GM-CSF, and/or one or more other cytokines in one or more previous treatment cycles and/or throughout treatment. In some embodiments, the patient has not received IL-2, GM-CSF, and/or one or more other cytokines in one or more previous treatment cycles and/or throughout treatment. In one embodiment, the antibody treatment period is not preceded, accompanied, and/or followed by one or more periods of IL-2, GM-CSF, and/or one or more other cytokines, i.e. the anti-GD2 antibody drug is administered to the patient without concomitant administration of IL-2, GM-CSF, and/or one or more other cytokines. In one embodiment, the period of treatment with a drug containing an anti-GD2 antibody is not accompanied by one or more periods of administration of cytokines (or drugs containing one or more cytokines), in particular not within the same treatment cycle and/or not within the same and the same total treatment time (including antibody administration). For example, the patient is not receiving granulocyte colony-stimulating factor (G-CSF), GM-CSF, IL-2, IL-12, and/or IL-15 at the same time. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient without simultaneous administration of IL-2. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient without simultaneous administration of GM-CSF. In one embodiment, the patient is not administered IL-2 and/or GM-CSF (or a formulation containing IL-2 and/or GM-CSF) within the same treatment cycle that includes the antibody administration period. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient without concomitant administration of IL-2 and/or GM-CSF. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient without simultaneous administration of a cytokine.

- 9 043975

A period of treatment with an anti-GD2 antibody-containing drug may be preceded, followed, and/or followed by one or more periods of treatment with a retinoid (or a retinoid-containing drug). In one embodiment, one or more periods of treatment with a drug containing an anti-GD2 antibody is followed by one or more periods of treatment with a retinoid. One or more periods of retinoid dosing may occur within or after the same or more treatment cycles comprising one or more periods of antibody administration, i.e. at the end of one or more treatment cycles including one or more antibody administration periods, in particular at the end of the last treatment cycle including one or more antibody administration periods, i.e. at the end of the total antibody treatment time. Accordingly, in one embodiment, there is a period of time between the period of administration of the antibody and the period of administration of the retinoid, which may be a period of time without treatment or may include one or more periods of administration of one or more other drugs or agents.

In one embodiment, one or more periods of treatment with a drug containing an anti-GD2 antibody is followed by one or more periods of treatment with a retinoid within the same treatment cycle. In one embodiment, the treatment cycle includes at least one period of treatment with a drug containing an anti-GD2 antibody and at least one period of treatment with a retinoid. In one embodiment, a treatment cycle may further include one or more non-treatment periods. In one embodiment, the treatment cycle includes at least one period of treatment with a drug containing an anti-GD2 antibody, followed by at least one period of treatment with a retinoid. In one embodiment, a treatment cycle may include at least one non-treatment period between at least one treatment period with an anti-GD2 antibody drug and at least one retinoid treatment period.

In one embodiment, the retinoid is retinoic acid (RA), for example, isotretinoin (cis-RA). The retinoid may be a first generation retinoid (e.g., retinol, retinal, tretinoin (retinoic acid, Retin-A), isotretinoin, or alitretinoin), a second generation retinoid (e.g., etretinate or its metabolite acitretin), and/or a third generation retinoid (e.g., tazarotene, bexarotene or adapalene). The retinoid may also be a retinoid derivative, in particular a synthetic retinoid derivative, such as fenretinide. Before or after one or more periods of treatment with a retinoid, there may be a period of treatment without administration of the antibody and/or IL-2 (or other cytokine).

Any such treatment period may be repeated. Any such period of treatment may be followed by an interval without treatment with the same and/or another drug or agent. In one embodiment, this period may be free of any treatment. In another embodiment, this interval is free of administration of the same drug or agent, however, other drugs or agents may be administered in this interval.

In addition, treatment according to the present invention may be preceded and/or accompanied by the administration of one or more analgesics (or a preparation containing one or more analgesics), such as, for example, non-steroidal anti-inflammatory drugs (NSAID, for example, indomethacin), and/or one or more several opioids and/or one or more other analgesics or combinations thereof. In one embodiment, the analgesic is an opioid, such as morphine and/or morphine derivatives, such as hydromorphone. Other opioids include, for example, tramadol, pethidine, codeine, pyritramide, levomethadone, as well as fentanyl, alfentanil, remifentanil and sufentanil.

As described above, the anti-GD2 antibody was typically administered to patients in combination with one or more cytokines (at conventional doses), especially IL-2 and/or GM-CSF. However, the combination of antibody administration with conventional cytokine administration increases the side effects of antibody use, in particular pain. Thus, the use of one or more analgesics, especially morphine, was required. And now, unexpectedly, it turns out that treatment with an anti-GD2 antibody is effective even without taking cytokines. Accordingly, if cytokines are completely eliminated, the side effect of pain (and other side effects) can be significantly reduced, so the administration of morphine and/or other analgesics can also be reduced. With the preparations and methods of the invention, it is possible to reduce the dose, change the route of administration (eg, from intravenous infusion to oral administration), shorten the period of use of the analgesics, and/or change the type of preparation of one or more analgesics. Thus, the present invention even allows outpatient treatment, at least for part of the treatment cycle, of patients receiving drugs containing an anti-GD2 antibody.

In one embodiment, with the administration regimen according to the invention, i.e. administration of an anti-GD2 antibody without coadministration of IL-2, GM-CSF and/or another cytokine, side effects are significantly reduced compared to a treatment regimen that includes concomitant administration of IL-2, GM-CSF and/or one or more other cytokines. In another embodiment, the side effect of pain is significantly reduced compared to a treatment regimen comprising concomitant administration of IL-2, GM-CSF and/or one or more other cytokines.

Accordingly, in one embodiment, the dose of one or more analgesics is reduced, especially

- 10 043975 benzomorphine, within the total treatment time, within one treatment cycle, during the period of antibody administration in one treatment cycle, from one day of antibody administration to the next day of antibody administration in one treatment cycle and/or from one treatment cycle to the next .

Doses of analgesics can be further reduced with long-term infusion of anti-GD2 antibody, for example, as a continuous IV infusion over 24 hours per day, as described in PCT/EP2012/061618 and PCT/EP2012/064970 and described here. Thus, the dose of analgesics can be significantly reduced with a treatment regimen of the invention without IL-2, and in some embodiments also without GM-CSF, and in some embodiments without any other cytokines, in combination with a continuous infusion of antibody. The term no other cytokines or no cytokines in the present invention means that neither IL-2 nor GM-CSF, nor any other cytokines are used, i.e. cytokines are not used at all for the appropriate treatment regimen and/or for a specific treatment time, for example, within the same treatment cycle and/or for the same total treatment time. Accordingly, in some embodiments, IL-2 is not used. In some embodiments, neither IL-2 nor GM-CSF is used. In some embodiments, no cytokines are used. Thus, the term without IL-2, and in some embodiments without GM-CSF, and in some embodiments without other cytokines, means the corresponding embodiments as described above, namely, treatment (e.g., treatment periods, treatment cycles, and/or overall treatment time) without IL-2, without IL-2 and without GM-CSF and/or without any cytokines.

One or more analgesics may be administered orally. One or more analgesics may also be administered as an intravenous infusion, especially as a continuous intravenous infusion over 24 hours per day. A period of use of one or more analgesics may precede and/or accompany a period of treatment with a drug containing an anti-GD2 antibody.

In some embodiments, one or more analgesics may be selected from GABA analogues, such as gabapentin. Accordingly, the patient may take gabapentin, for example, three days before the start of the antibody administration period.

The following dosages and examples of analgesic use are typical dosages that may be reduced as described herein. Typically, gabapentin is administered orally at a dose of 10 mg/kg one, two or three times a day. Gabapentin can be administered at doses up to 300 mg/kg. Gabapentin is available and can be administered as an oral solution containing 250 mg/5 mL gabapentin or in capsules (100 mg, 300 mg, and 400 mg). The use of gabapentin may be used instead of or in addition to the use of morphine and/or other analgesics. In addition, patients may receive paracetamol (10 to 15 mg/kg every 4 hours or four times daily, orally or intravenously), ibuprofen (5 to 10 mg/kg orally every 6 to 12 hours) ), metamizole (at a dose of 10 to 15 mg/kg orally every 4 hours), diphenhydramine (at a dose of 0.5 to 1 mg/kg orally or intravenously), and/or indomethacin (for example, at a dose of 0.3 up to 0.5 mg/kg or 25 or 50 mg, orally or intravenously every 6 hours). Such use of paracetamol, ibuprofen, metamizole and/or indomethacin may be used instead of or in addition to the use of morphine and/or gabapentin and/or other analgesics.

In some embodiments, morphine is used as an analgesic, optionally in combination with one or more other analgesics. As an example in table. 1 shows some of the usual doses of morphine that are administered before and/or during the period of treatment with a drug containing an anti-GD2 antibody. In this typical dose example, ch14.18/CHO (APN311) is administered as an infusion 8 hours per day for 5 days at a dose of 20 mg/m ² /day, i.e. 100 mg/m ² /cycle, IL-2 is administered subcutaneously at 6 mIU/m ² /day for 5 days, twice per cycle (i.e. 60 mIU/m ² /cycle), for 5 cycles , and morphine hydrochloride is administered every antibody infusion day (i.e., every day in the treatment cycle when the patient is administered a drug containing anti-GD2 antibody) as a bolus of 0.05 mg/kg/h for 2 hours before the start of the APN311 infusion. , with an infusion rate of 0.03 mg/kg/h over 8 hours during APN311 infusion, and in between with an infusion rate of 0.01 mg/kg/h over 14 hours on the first day of treatment with APN311 and 4 hours each subsequent days of treatment, if well tolerated (with an interval of 10 hours without administration of morphine). The dose is increased (eg, the infusion rate is increased during antibody infusion) and/or additional bolus doses are given as needed. Accordingly, the prescribed dose of morphine is at least 0.38 mg/kg per day, at least 2 mg/kg per treatment cycle (including 5 days of antibody administration), and at least 10 mg/kg over the entire treatment period (including three cycle).

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Table 1

Morphine infusion schedule

Duration of morphine infusion (hours) Morphine infusion rate (mg/kg/h) Morphine dose (mg/kg) Prev. infusion 2 0.05 OD Infusion during APN311 administration 8 0.03 0.24 Intermediate infusion 14 or 4 0.01 0.14 or 0.04 Total dose for 1 day of treatment (mg/kg/24h) 0.48 or 0.38

Prepare a solution of 10 mg of morphine in 40 ml of 5% glucose (0.25 mg in 1 ml).

In another example of typical doses, APN311 is administered as an infusion 8 hours per day for 5 days for 3 cycles at a dose of 10, 20 or 30 mg/m ² /day, i.e. 50, 100, 150 mg/ ^m2 /cycle, IL-2 is administered subcutaneously at 6 mIU/ ^m2 /day for 5 days, twice per cycle (i.e. 60 mIU/ ^m2 /cycle), and morphine hydrochloride is administered every day with the antibody bolus at a dose of 0.5-1.0 mg/kg (just before the start of the antibody infusion) and as a continuous infusion at a rate of 0.05 mg/kg/h during the APN311 infusion. The dose is increased (eg, the infusion rate is increased during antibody infusion) and/or additional bolus doses are given as needed. Accordingly, the prescribed dose of morphine (or usual dose of morphine) is at least 0.9 mg/kg per day, at least 4.5 mg/kg per treatment cycle (including 5 days of antibody administration), and at least 13.5 mg/kg for the entire treatment period (including three cycles).

In other examples of conventional treatment with ch14.18 and one or more cytokines, morphine is administered by infusion at rates up to 1.2 mg/kg/hour over 24 hours.

In another example of typical doses, ch14.18/Sp2/0 is administered at a dose of 25 mg/m ² /day for 4 days. In one embodiment, each dose of ch14.18/Sp2/0 is administered by IV infusion over 5.75 hours or more (can be extended to 20 hours) or 10 hours or more (can be extended to 20 hours), starting at 1. 25 mg/ ^m2 /hx0.5 h, then 2.5 mg/ ^m2 /hx0.5 h, and optionally with a further increase in the infusion rate to 3.75 mg/ ^m2 /hx0.5 h, and then up to 5 mg/m ² /h for the remaining dose if well tolerated. IL-2 is administered every second cycle (for example, cycles 2 and 4) as a continuous IV infusion over 4 days (96 hours) at 3 mIU/m ² /day in the 1st week of the treatment cycle and 4.5 mIU/m ² /day in the 2nd week of the treatment cycle (i.e. 30 mIU/m ² /cycle). GM-CSF is administered s.c. at a daily dose of 250 mcg/ ^m2 . In one embodiment, each treatment cycle begins on day 0, with day 0 of the treatment cycle being the first day of administration of the corresponding cytokine. The total treatment time may include 1, 2, 3, 4 or 5 or more treatment cycles. In table Figures 2, 3 and 4 provide examples of common treatment regimens.

table 2

Conventional 5-cycle dosing regimen for ch14.18/Sp2/0, cytokines, and isotretinoin (retinoic acid or RA)

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 chl4.18 chl4.18 chl4.18 chl4.18 chl4.18 GM-CSF aldezleukin (IL-2) GM-CSF aldezleukin (IL-2) GM-CSF R.A. R.A. R.A. R.A. R.A.

In this example of usual doses, ch14.18/SP2/0 is administered every 28 days at 25 mg/m ² /day x 4 days for all 5 cycles; GM-CSF 250 mcg/ ^m2 /day for 14 days; aldezleukin (IL-2) 3 mIU/m ² /day during the 1st week and 4.5 mIU/m ² /day during the 2nd week.

Table 3

day of the first cycle, but in accordance with the scheme on the 10th day of the third and fifth cycle. Accordingly, in contrast to the above treatment regimen, the 24th day of the first treatment cycle is also the last day of RA administration in the first cycle.

In this example of typical doses, GM-CSF is administered at 250 mcg/ ^m2 /day as a subcutaneous injection (highly recommended) or as an IV infusion 2 hours per day from day 0 to day 13 (daily infusion ch14 .18/SP2/0 and for 3 days before and 7 days after the administration of the antibody).

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Table 4

Scheme of treatment cycles with aldezleukin (IL-2)

From the 28th to the 31st day of cycles with aldezleukin, no treatment is carried out. On day 32, the next treatment cycle (with GM-CSF) begins (day 32=day 0 of the next cycle).

As an example of typical dosages, aldezleukin (interleukin-2, IL-2) may be administered at a dose of 3 mIU/m ² /day by continuous infusion (using a CADD® ambulatory infusion pump or similar infusion pump) for 4 days (days 0- 3) in the first week of each cycle. In the second week of each cycle, aldezleukin (IL-2) can be administered at 4.5 mIU/m ² /day for 4 days (days 7-10, with infusion ch14.18/SP2/0). Aldezleukin can be infused IV continuously over 96 hours through a catheter using an ambulatory infusion pump in 5% dextrose in water (may contain 0.1% human serum albumin if desired), and the total volume depends on the pump.

A sixth treatment cycle of 14 days without treatment (starting on day 24 of the fifth cycle, which will be day 0 of the sixth cycle) may be added, followed by 14 days of isotretinoin alone.

In one example of common dosages in these conditions (i.e., those procedures described above), hydroxyzine (1 mg/kg; max, 50 mg dose) or diphenhydramine (0.5-1.0 mg/kg; max , dose 50 mg) is administered intravenously over 10 minutes, starting 20 minutes before the infusion ch14.18/SP2/0; acetaminophen (10 mg/kg; max dose 650 mg) administered po 20 min before infusion ch14.18/SP2/0; and/or morphine sulfate is administered at a loading dose of 50 mcg/kg immediately before the administration of ch14.18/SP2/0, and then continued through the IV at an infusion rate of 20-50 mcg/kg/h for an additional 2 hours after completion of the ch14 infusion. 18/SP2/0. In addition, other narcotics, such as hydromorphone or fentanyl, can be used. Alternatively, lidocaine infusion can be used in combination with an IV bolus of morphine if needed. Recommendations for administering lidocaine by infusion are given below.

Administration of lidocaine (in usual doses):

a) administer lidocaine IV as a bolus of 2 mg/kg in 50 cm ³ of standard saline (NS) over 30 minutes before the start of the infusion ch14.18/SP2/0;

b) at the beginning of the ch14.18/SP2/0 infusion, begin an IV infusion of lidocaine at a dose of 1 mg/kg/h and continue for another 2 hours after completion of the ch14.18/SP2/0 infusion;

c) IV morphine can be given as a bolus of 25-50 mcg/kg every 2 hours if needed.

In this example of conventional dosing, one could also consider administering gabapentin with loading doses of morphine and administering morphine by infusion/bolus as needed; may begin with 10 mg/kg/day gabapentin and titrate up to 30-60 mg/kg/day depending on clinical response.

In this example of usual doses, hydroxyzine (or diphenhydramine) and acetaminophen can be repeated every 6 hours if necessary; IV or per os.

In this example of normal dosing, additional doses of morphine may be given during the ch14.18/SP2/0 infusion to treat neuropathic pain followed by increasing the rate of morphine sulfate infusion, but patients must be monitored closely. If patients cannot tolerate morphine (eg, itching), fentanyl or hydromorphone may be substituted for morphine. Alternatively, lidocaine infusion can be used in conjunction with an IV morphine bolus if necessary.

The term morphine dose in the present invention means the amount of morphine (in mg or μg) per kg of body weight of the patient. Accordingly, if it refers to the daily dose of morphine, then it means the amount of morphine (in mg or mcg) per kg of the patient's body weight per day, and if it refers to the dose of morphine per hour, then it means the amount of morphine (in mg or mcg) per kg of patient's body weight per hour (or morphine infusion rate), and if this refers to the dose of morphine per treatment cycle, then this means the amount of morphine (in mg or mcg) per kg of patient's body weight per treatment cycle, and if this refers to to the dose of morphine for the entire treatment period, this means the amount of morphine (in mg or mcg) per kg of the patient’s body weight for the entire treatment period.

In some embodiments, morphine and/or other analgesics may be administered at the usual daily dose, but with a reduced frequency of administration, for example, only every other day, if previously it was administered every day. In another example, in which morphine and/or other analgesics were typically administered as a continuous IV infusion over several days, either the infusion time per day (receiving an intermittent infusion) or the total duration of the continuous infusion could be reduced, i.e. number of days of continuous infusion.

In some embodiments, the daily dose of one or more analgesics on one or more days of the antibody administration (i.e., those days of the treatment cycle on which the anti-GD2 antibody-containing drug is administered to the patient) in the treatment cycle of the invention (without IL-2 and in some embodiments without IL-2 and without GM-CSF, and in some embodiments without cytokine treatment at all) will be lower than the typical daily dose administered on one or more days of antibody administration in a conventional treatment cycle (with conventional cytokine treatment).

- 13 043975

In some embodiments, the dose (e.g., daily dose) of one or more analgesics (e.g., morphine) is reduced over time, e.g., within the total treatment time, within one treatment cycle, during the antibody administration period within one treatment cycle, from one day administration of the antibody or analgesic to the next day of administration of the antibody or analgesic in one treatment cycle and/or from one treatment cycle to the next. In some embodiments, the dose of the analgesic, particularly the dose of morphine, is continuously reduced within one treatment cycle, during the period of antibody administration in one treatment cycle and/or from one day of antibody or analgesic/morphine administration to the next day of antibody or analgesic/morphine administration in within one treatment cycle.

In some embodiments, a daily dose of morphine administered over one or more days of antibody infusion regimen without IL-2, and in some embodiments without GM-CSF (and without IL-2), and in some embodiments without any other cytokines in accordance with the invention, and/or the dose of morphine on all days of administration of the antibody or morphine will be lower than the daily dose of morphine when administering the antibody with the usual doses of one or more cytokines. In some embodiments, the daily dose of morphine or the dose of morphine per cycle administered in an antibody infusion regimen without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any cytokines, is 90% or less, 80%. or less, 70% or less, 60% or less, or 50% or less, or 40% or less, or 30% or less, or 20% or less, or 10% or less of the morphine dose administered or prescribed antibody infusions with regular doses of cytokines. In some embodiments, a daily dose of morphine administered over one or more days of continuous intravenous infusion of an antibody without IL-2, and in some embodiments also without GM-CSF, and in some embodiments also without any other cytokines in accordance with the invention, and /or on all days of antibody or morphine administration is less than 0.9, 0.72, 0.48, 0.38, 0.4375 and/or 0.205 mg/kg/day.

In some embodiments, morphine is administered only on some but not all days on which the antibody is administered, such as only on the first 1, 2, 3, 4, 5, 6, or 7 days of antibody administration without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any other cytokines. In some embodiments, morphine is not administered on one or more days during antibody management or even throughout the entire treatment cycle. In some embodiments, morphine is administered in only some, but not all, treatment cycles.

In some embodiments, the rate of morphine infusion, i.e. the amount of morphine per kg of patient body weight (or morphine dose) per hour administered over one or more hours or days of intravenous antibody infusion without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any other cytokines in accordance with the invention and/or at all hours or days of morphine administration will be lower than the standard morphine infusion rate prescribed in the regular cytokine dose regimen and/or lower than the morphine infusion rate in the normal cytokine dose examples described higher. In some embodiments, the rate of infusion of morphine administered over one or more days of intravenous infusion of an antibody without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any other cytokines in accordance with the invention, and/or on all days of antibody or morphine administration is less than 50, 40, 30, 20, 10 and/or 5 mcg/kg/h and/or less than any interval between these infusion rates. In some embodiments, the rate of infusion of morphine administered over one or more days of intravenous antibody infusion without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any other cytokines in accordance with the invention and/or all days of antibody or morphine administration is less than 50 mcg/kg/h in the first and optionally in subsequent treatment cycles, less than 40 mcg/kg/h in the second and optionally in subsequent treatment cycles, less than 30 mcg/kg/h in the third and optionally in subsequent treatment cycles, less than 20 mcg/kg/h in the fourth and optionally in subsequent treatment cycles and/or less than 10 mcg/kg/h in the fifth and optionally in subsequent treatment cycles.

In some embodiments, a dose of morphine per treatment cycle administered during one or more treatment cycles comprising intravenous infusion of an antibody without IL-2, in some embodiments without GM-CSF, and in some embodiments without any other cytokines in accordance with the invention will be lower than the morphine dose per treatment cycle of a conventional cytokine infusion regimen, for example, 90, 80, 70% or less in the first treatment cycle; 80, 70, 60% or less in the second cycle of treatment; 60, 50, 40% or less in the third cycle of treatment; 45, 35, 25% or less in the fourth cycle of treatment; and/or 30%, 20%, 10% or less in the fifth treatment cycle. In some embodiments, the dose of morphine per treatment cycle in the second and subsequent treatment cycles administered during one or more treatment cycles comprising intravenous infusion of an antibody without IL-2, in some embodiments without GM-CSF, and in some embodiments without any other cytokines in accordance with the invention will be lower than the dose of morphine per cycle of treatment according to the antibody infusion regimen with conventional doses of cytokines. In some embodiments, the dose of morphine in a given treatment cycle and in subsequent treatment cycles and/or throughout the treatment period will be lower than the dose of morphine per treatment cycle using a conventional infusion schedule. In some embodiments, the dose of morphine per treatment cycle administered during one or more treatment cycles comprising intravenous infusion of the antibody without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any other

- 14 043975 cytokines in accordance with the invention is less than 7.2, 4.8, 4.5, 2, 1.75 and/or 0.82 mg/kg/cycle or less than at any interval between these doses.

In some embodiments, the dose of morphine over the entire treatment period of an infusion regimen of the invention (i.e., an intravenous infusion of an antibody without IL-2, in some embodiments without GM-CSF, and in some embodiments without any other cytokines of the invention ) will be lower than the morphine dose for the entire treatment period using a conventional infusion regimen (i.e., an antibody infusion regimen with normal doses of cytokines). In one embodiment, the morphine dose over the entire treatment period of the infusion regimen according to the invention is 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less, 40% or less, 30% or less, 20% or less, 10% or less of the morphine dose during the entire treatment period according to the regular infusion schedule, and/or less than at any time between these doses. In some embodiments, the dose of morphine for the entire treatment period of the infusion regimen according to the invention without administration of cytokines is less than 43.2, 28.8, 13.5, 10, 8.75 and/or 4.1 mg/kg for the entire treatment period and /or less than in any interval between these doses.

In some embodiments, the standard doses of morphine for conventional infusion regimens provided herein in comparison with the doses of morphine for infusion regimens of the present invention refer to the standard doses of morphine for a given regimen or to the doses of morphine prescribed for a given regimen (for example, those given in clinical trial protocols ). In some embodiments, unit dosages of morphine provided herein refer to the dose of morphine that is administered on the first day of treatment with an anti-GD2 antibody preparation in a treatment cycle of the antibody infusion regimen of the invention and/or the conventional regimen and is referred to as the initial morphine dose.

Accordingly, the term morphine unit dose as used herein includes both morphine doses in treatment regimens other than those of the present invention (eg, with cytokine administration) and/or initial doses of morphine (eg, in treatment regimens with or without cytokine administration); and covers all examples of such morphine doses given herein in comparison with other morphine doses in antibody infusion regimens without IL-2, and in some embodiments without IL-2 and without GM-CSF, and in some embodiments without any cytokines according to with the present invention.

In some embodiments, the unit dose of morphine per 1 hour infusion, i.e. The standard infusion rate during antibody administration is 50 mcg/kg/h. In some embodiments, the unit dose of morphine per 1 hour infusion, i.e. The standard infusion rate during antibody administration is 30 mcg/kg/h. In some embodiments, the unit dose of morphine is 50, 40, 30 and/or 20 μg/kg/hour. In some embodiments, the unit dosage of morphine is 0.9, 0.72, 0.48, 0.38, 0.4375, and/or 0.205 mg/kg/day. In some embodiments, the unit dose of morphine is 7.2, 4.8, 4.5, 2, 1.75 and/or 0.82 mg/kg/cycle. In some embodiments, the unit dosage of morphine is 43.2, 28.8, 13.5, 10, 8.75, and/or 4.1 mg/kg over the entire treatment period. In some embodiments, the unit dose of indomethacin is 0.3 to 0.5 mg/kg, or 25 or 50 mg per dose, orally or intravenously every 6 hours. In some embodiments, unit doses of morphine in conventional infusion schedules (with cytokine administration) are given higher compared to the morphine doses of the infusion regimens according to the invention (without the administration of cytokines), refer to the doses of morphine that are actually administered to patients (for example, the corresponding average values of morphine doses administered to all treated patients in the same group when treated in the same same conditions).

The morphine dosages for the infusion regimens of the invention given here in comparison with the morphine doses for conventional infusion regimens may refer to the reduced standard morphine doses for a given regimen according to the invention or to the reduced doses of morphine prescribed for a given regimen (for example, those given in clinical trial protocols ). In some embodiments, a reduced dose of morphine according to the infusion schedule according to the invention per 1 hour infusion, i.e. the infusion rate over one or more hours or days of administration of an antibody of the invention without IL-2, and in some embodiments without GM-CSF, and in some embodiments without any cytokines, is less than 50 μg/kg/hour). In some embodiments, the dose of morphine per 1 hour infusion, i.e. the infusion rate over one or more hours or days of administration of the antibody of the invention is less than 30 μg/kg/hour. In some embodiments, the morphine dosages for the infusion regimens of the invention given above in comparison with the morphine dosages for conventional infusion regimens refer to the morphine doses that are actually administered to patients (for example, corresponding to the average morphine doses administered to all treated patients in the same group at treatment under the same conditions).

In general, individual doses of analgesics may vary depending on the individual pain tolerance of patients. The dosage can be adjusted to obtain optimal analgesia.

A period of treatment with an anti-GD2 antibody drug may be combined with one or more periods of taking a retinoid, one or more periods of taking an analgesic, one or more periods of taking other drugs or agents (except taking cytokines), and/or one or more periods of no treatment . In one embodiment, a period of treatment with an anti-GD2 antibody-containing drug is combined with one or more

- 15 043975 with other treatment periods, constituting one treatment cycle. Accordingly, in one embodiment, a treatment cycle includes a period of treatment with a drug containing an anti-GD2 antibody, and optionally one or more periods of administration of other drugs, agents and/or procedures (except administration of cytokines). Any such treatment periods within the same treatment cycle may partially and/or completely overlap, as further described herein.

In one embodiment, the patient who receives the antibody also receives a retinoid (eg, isotretinoin) and optionally morphine and/or one or more morphine derivatives and/or one or more other analgesics. In one embodiment, the period of treatment with the anti-GD2 antibody-containing drug is not preceded and/or followed by a period of administration of one or more cytokines. In one embodiment, the period of treatment with a drug containing an anti-GD2 antibody is not accompanied by a period of taking morphine and/or one or more analgesics. In one embodiment, the period of treatment with a drug containing an anti-GD2 antibody is not accompanied by a period of administration of one or more cytokines. In one embodiment, the period of treatment with the anti-GD2 antibody drug is not preceded and/or followed by a period of administration of one or more cytokines and is not accompanied by a period of administration of one or more cytokines.

In one embodiment, one treatment cycle includes from 28 to 49 days, such as 28, 35, 42 or 49 days, or any interval between these periods. A treatment cycle begins on the day the patient first receives any of the treatments contained in that cycle (and may be referred to as day 0 or day 1), such as administration of a drug containing an anti-GD2 antibody and/or any other drug or treatment (beyond except for taking cytokines).

The period of administration of the anti-GD2 antibody may be preceded, followed, and/or followed by a period of administration of a retinoid (eg, isotretinoin) either directly or at intervals of one or more days without treatment, for example 1, 2, 3, 4 or 5 days without treatment. In one embodiment, a retinoid (eg, isotretinoin) is administered orally twice daily at a dose of 160 mg/ ^m2 /day (in equal doses, ie, 2x80 mg/ ^m2 ). In one embodiment, the retinoid (eg, isotretinoin) is administered over a period of 14 days, eg, from days 1 to 14 of a treatment cycle or from days 19 to 32 of a treatment cycle. A period of taking a retinoid (eg, isotretinoin) may be followed by an interval of one or more days without treatment, such as 1, 2, 3, 4, or 5 days without treatment.

In one embodiment, the treatment cycle includes one 14-day period of taking a retinoid (eg, isotretinoin), for example, from days 1 to 14 of the treatment cycle, followed by 7 days without treatment (days 15-21 of the treatment cycle), and a 5-day period administration of anti-GD2 antibody (eg, infusion at 20 mg/m ² /day over 8 hours with a dose of 100 mg/m ² /cycle), for example from 22 to 26 days of the treatment cycle, after which the next cycle begins on 29- 1st day, which becomes the 1st day of the next treatment cycle. In other embodiments, the treatment cycle includes one 14-day period of dosing with a retinoid (e.g., isotretinoin), such as days 1 to 14 of the treatment cycle, followed by one 5-day period of anti-GD2 antibody administration (e.g., infusion at 20 mg/day). m ² /day for 8 hours with a dose of 100 mg/m ² /cycle), for example from 22 to 28 days of the treatment cycle, after which the next cycle begins on the 29th day, which becomes the 1st day of the next treatment cycle .

In one embodiment, the treatment cycle includes one 10-day period of anti-GD2 antibody administration (eg, at 10 or 15 mg/m ² /day with a dose of 100 or 150 mg/m ² /cycle), for example from 8 to 17 day of the treatment cycle, and one 14-day period of taking a retinoid (eg, isotretinoin), for example from days 19 to 32 of the treatment cycle, followed by 3 days without treatment, after which the next cycle begins on day 36, which becomes 1- m day of the second treatment cycle.

In one embodiment, the treatment cycle includes one 14-day period of anti-GD2 antibody administration (eg, at 7 or 15 mg/m ² /day with a dose of 100 or 210 mg/m ² /cycle), for example from 8 to 21 day of the treatment cycle, and one 14-day period of taking a retinoid (eg, isotretinoin), for example from days 26 to 39 of the treatment cycle, followed by 3 days without treatment, after which the next cycle begins on day 43, which becomes 1- m day of the second treatment cycle.

In one embodiment, the treatment cycle includes one 15-day period of anti-GD2 antibody administration (e.g., at 10 mg/m ² /day with a dose of 150 mg/m ² /cycle), for example, from days 8 to 22 of the treatment cycle, and one 14-day period of taking a retinoid (eg, isotretinoin), for example from days 26 to 39 of the treatment cycle, followed by 3 days without treatment, followed by the next cycle on day 43, which becomes day 1 of the second cycle treatment.

In one embodiment, the treatment cycle includes one 21-day period of anti-GD2 antibody administration (eg, at 7 or 10 mg/m ² /day with a dose of 150 or 210 mg/m ² /cycle), for example from 8 to 28 day of the treatment cycle, and one 14-day period of taking a retinoid (eg, isotretinoin), for example from days 33 to 46 of the treatment cycle, followed by 3 days without treatment, after which the next cycle begins on day 50, which becomes 1- m day of the second treatment cycle.

In one embodiment, the treatment cycle includes one 4-day period of treatment with a drug containing an anti-GD2 antibody (for example, ch14.18/SP2/0), for example, when administered at a dose of 25, 20, 17.5 or 15 mg/day. m ² /day, for example from 3 to 6 days of a 24-day treatment cycle, starting from day 0, or from 7 to 10 days of a 32-day treatment cycle, starting from day 0, for example, for 5 cycles; and one period of RA administration,

- 16 043975 for example isotretinoin, from 10 to 23 days of the treatment cycle, starting from day 0, or from 14 to 27 days of the treatment cycle, starting from day 0. In one embodiment, the treatment regimen is the same as shown in table. 2, 3 and/or 4, but without IL-2, and in some embodiments without IL-2 and without GM-CSF, and in some embodiments without any cytokines at all. Accordingly, in one embodiment, the treatment regimen is the same as shown in table. 2, 3 and/or 4, but no cytokines at all.

The treatment cycle may be repeated either in the same form or in a modified form, for example, with a different dose or schedule, or with other additional treatments (for example, with one or more other analgesics). Thus, the total treatment time (eg, the period of time including all successive treatment cycles, or the entire period of continuous treatment) may be at least 1 or 2 or more cycles, or 10 or more cycles. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient over two or more treatment cycles. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient as a continuous infusion over two or more treatment cycles. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient as a continuous infusion over two or more treatment cycles without simultaneous administration of IL-2. In one embodiment, a drug containing an anti-GD2 antibody is administered to a patient as a continuous infusion without concomitant administration of IL-2 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19 or 20 cycles or more. In one embodiment, the total treatment time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles or more. The total treatment time can even be up to 20 or more treatment cycles. As described above, treatment cycles may include non-treatment periods (intervals in which the patient does not receive any treatment, ie, no antibody, cytokine, or any other drug). Thus, in the present invention, the total treatment time may also include such non-treatment intervals within treatment cycles.

In one embodiment, the 35-, 42-, or 49-day treatment cycle as defined above is repeated at least 4 or 5 times, so that the entire continuous treatment period is at least 5 or 6 treatment cycles.

The effect of using an antibody without one or more cytokines can be determined by a complement dependent cytolysis (CDC) assay or a whole blood test (WBT). WBT is a technique in which target cells or target components (ie, cells, liposomes, or other cell-like compartments to be lysed) are contacted with appropriately anticoagulated whole blood from a patient. The CDC method may be, for example, a standard CDC method known in the art (for example, as described in Indusogie et al., J. Immunol., 2000, 164(8):4178-84; Zeng et al., Molecular Immunology, 2005, 42(11):1311-9; or in WO 2005/070967). The CDC and/or WBT method can be performed on GD2-positive cells of the target cell type of the cancer cell line of the GD2-positive cancer being treated. For example, if the patient to be treated has neuroblastoma, the cell line may be a neuroblastoma cell line, such as human neuroblastoma LAN-1 cells. In another example, if the patient to be treated has melanoma, the cell line may be a melanoma cell line, such as human M21 melanoma cells. In yet another example, the target cells in the CDC and/or WBT assay are patient-derived tumor cells, i.e. autologous tumor cells of the patient. In another embodiment, the target component of the CDC and/or WBT assay is liposomes bearing GD2 on the surface. The target cells or target components are labeled with a signaling component, for example a radioactive component such as ⁵¹ Cr or a fluorescent component such as calcein. The signaling component is contained in the target cells or target components, i.e. within target cells or a target component (eg, liposomes filled with a signal component and displaying GD2 on the surface), and is released upon lysis of the target cells or target components. Thus, the signal component provides reading of the analysis results. Cells or target components loaded with a signaling compound are contacted with whole blood, serum or plasma in a certain ratio. Whole blood, plasma or serum for CDC or WBT may be diluted, for example, at a ratio of 1:2 or higher, such as 1:4, 1:5 or 1:10, or anywhere in between before adding it to samples . However, they can also be added undiluted to samples. The final concentration of whole blood, plasma or serum in CDC or WBT samples may be, for example, 10 to 50%. Lysis of target cells or target components can be measured by the release of the specified signal component in a scintillation counter or by spectrophotometry. For example, lysis of target cells or target components can be measured by measuring the amount of ⁵¹ Cr released into the supernatant in a scintillation counter. The percentage of lysis can be determined using the following equation:

100x(experimental release-spontaneous release)/(maximum releasespontaneous release)

For the CDC assay, cytolytic components (or effector components) are provided by serum or appropriately anticoagulated plasma obtained from the patient or

- 17 043975 donor and containing components of the complement system. For the WBT method, the cytolytic components (or effector components) are provided by appropriately anticoagulated whole blood obtained from the patient or donor and containing the components of the complement system, as well as all cellular components, as well as any other components contained in the whole blood that might be relevant to the lysis of target cells, as well as the interaction of all components (for example, it is known that complement activation activates certain effector cells such as granulocytes). For CDC and/or WBT, serum, plasma or whole blood can be added to target cells or target components at various dilutions.

In addition, anti-GD2 antibody can be added to one or more CDC and/or WBT assay samples at varying concentrations, for example, to generate a standard curve.

In another embodiment, one or more anti-idiotypic (anti-Id) anti-GD2 antibodies recognizing the variable domain of anti-GD2 antibodies can be added to samples to inhibit antibody-mediated lysis of target cells, for example, as a negative control or to test the specificity of the assay and whether that lysis of target cells, measured in the absence of anti-Id antibodies, is antibody mediated or antibody dependent.

In some embodiments, the increased level of cytolysis after the first one or more days of antibody administration (compared to the level of cytolysis before antibody administration) persists throughout the entire treatment cycle. In some embodiments, this increased level of cytolysis persists throughout the duration of treatment, i.e. even during periods of time when the patient is not receiving a drug containing an anti-GD2 antibody, i.e. in the intervals between periods of treatment with a drug containing an anti-GD2 antibody (if any, i.e. if the patient does not continuously receive a drug containing an anti-GD2 antibody throughout the entire treatment period).

In one embodiment, the level of cytolysis (the level of target cell lysis, e.g., as measured by the CDC or WBT method) in patient blood samples treated in accordance with the present invention is increased compared to the level of cytolysis before the first period of treatment with the antibody preparation, throughout the period of time starting from the end of the first antibody administration period to the end of the last treatment cycle, i.e. even between periods of antibody administration. In one embodiment, the level of cytolysis is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or anywhere in between, for the entire period of time beginning at the end of the first period. administration of the antibody until the end of the last treatment cycle. In one embodiment, the level of cytolysis after the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth period of anti-GD2 antibody administration is at least 10, 20, 30, 40, 50, 60, 70. 80, 90 or 100% or anywhere in between. In one embodiment, the level of cytolysis before the second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth period of anti-GD2 antibody administration is at least 10, 20, 30, 40, 50, 60, 70, 80. 90 or 100% or anywhere in between.

Examples

Example 1: APN311 sequences and associated data.

APN311 sequence data.

Table 5

Molecular weight (mw) and pI (calculated)

^ρΐυ MW [D] ^υ Quantity a. To. Conditions 2D-DIGE ²⁾ Antibody 8.61 144701.10 1324 will not restore. X Antibody (½) 8.58 72359.56 662 will restore. X Heavy chain 8.58 48306.59 442 will restore. X Light chain 8.48 24070.98 220 will restore. X

1) Calculated via http://web.expasy,org/compute_pi/.

2) Due to the molecular weight of the dyes, a shift towards a slight increase in molecular weight should be expected with 2D-DIGE.

Nucleotide sequence (cDNA, including leader).

TAG acts as a stop codon and is therefore not translated into a peptide sequence.

- 18 043975

Light chain (SEQ ID NO: 1).

lts sll sss sss barely stt sts tts sts sts stl sts tss sts slt slt forest let 61 GAA ATA GTG ATG ACG CAG TST CCA GCC ACC CTG TST GTG TST CCA GGG GAA AGA GCC ACC 121 STS TSS TGC AGA TST AGT CAG AGT CTT GTA CAC CGT AAT GGA AAC ACC TAT TTA CAT TGG 181 TAC CTG CAG AAG CCA GGC CAG TCT CCA AAG CTC CTG ATT CAC AAA GTT TCC AAC CGA TTT 241 TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ASA STS AAG ATS 301 AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TAT TTC TGT TCT CAA AGT ASA CAT GTT CCT 361 CCG CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGA ACT GTG GCT GCA CCA TCT 421 GTC TTC ATS TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC 481 CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC 541 CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC 601 CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC TGC 661 GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT 721 TAG

Heavy chain (SEQ ID NO: 2).

ATG GGA TGG ACC TGG ATC TTT ATT TTL ATC CTG TCG GTA ACT LSL GGT GTC CAC TCT GAG 61 GTC CAA CTG CTG CAG TCT GGA CCT GAG CTG GAG AAG CCT GGC GCT TCA GTG ATG ATA TCC 121 TGC AAG GCT TCT GGT TCC TCA TTC ACT GGC TAC AAC ATG AAC TGG GTG AGG CAG AAC ATT 181 GGA AAG AGC CTT GAA TGG ATT GGA GCT ATT GAT CCT TAC TAT GGT GGA ACT AGC TAC AAC 2 41 CAG AAG TTC AAG GGC AGG GCC ACA TTG ACT GTA GAC AAA TCG TCC AGC ACA GCC TAC ATG 3 01 CAC CTC AAG AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GTA AGC GGA ATG GAG 361 TAC TGG GGT CAA GGA ACC TCA GTC ACC GTC TCC TCA GCC TCC ACC AAG GGC CCA TCG GTC 421 TTC CCC CTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG 481 GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC AGC 541 GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGC GTG 601 GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG 661 CCC AGC AAC ACC AAG GTG GAC AAG AGA GTT GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA 721 TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA 781 AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC 841 GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT 901 AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC 961 CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC 1021 AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA 1081 CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG 1141 ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG 1201 CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC 1261 CTC TAT AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC 1321 TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCC CCG 1381 GGT AAA TGA

Nucleotides 1-60 (crossed out): leader sequence.

Last nucleotides (crossed out): stop codon.

Peptide sequence (incl. signal peptide).

The signal peptide is cleaved off during post-translational processing and is no longer part of the final recombinant protein.

- 19 043975

Light chain (SEQ ID NO: 3).

Μ Ε .APAQLLFLLLL'W L Ρ D Τ Τ G

EIVMTQSPATLSVSPGERAT

LSCRSSQSLVHRNGNTYLHW

YLQKPGQSPKLLIHKVSNRF

SGVPDRFSGSGSGTDFTLKI

101 SRVEAEDLGVY FCSQSTHVP

121 PLT FGAGTKLELKRTVAAPS

141 VFI FPPSDEQLKSGTASVVC

161 LLNNFYPREAKVQWKVDNAL

181 QSGNSQESVTEQDSKDSTYS

201 LSSTLTLSKADYEKHKVYAC

221 EVTHQGLSSPVTKSFNRGEC

Heavy chain (SEQ ID NO: 4).

MGWTWIFILILSVTTGVHSE

VQLLQSGPELEKPGASVMIS

CKASGSSFTGYNMNWVRQNI

GKSLEWIGAIDPYYGGTSYN

QKFKGRATLTTVDKSSSSTAYM

101 HLKSLTSEDSAVYYCVSGME

121 YWGQGTSVTVSSASTKGPSV

141 FPLAPSSKSTSGGTAALGCL

161 VKDYFPEPVTVSWNSGALTS

181 GVHTFPAVLQSSGLYSLSSV

201 VTVPSSSLGTQTYICNVNHK

221 PSNTKVDKRVEPKSCDKTHT

241 CPPCPAPELLGGPSVFLFPP

261 KPKDTLMI SRTPEVTCVVVD

281 VSHEDPEVKFNWYVDGVEVH

301 NAKTKPREEQYNSTYRVVSV

321 LTVLHQDWLNGKEYKCKVSN

341 KALPAPIEKTISKAKGQPRE

361 PQVYTLPPSREEMTKNQVSL

381 TCLVKGFYPSDIAVEWESNG

401 QPENNYKTPPPVLDSDGSFF

421 LYSKLTVDKSRWQQGNVFSC

441 SVMHEALHNHYTQKSLSLSP

461 G K

Amino acids 1-20 (crossed out): leader sequence.

Two GMP-compliant batches of the ch14.18/CHO antibody (APN311) were produced. The two batches of the drug that were produced were: batch T651204-A (containing 4.3 ml of antibody (4.6 mg/ml)) and batch T900310-A (containing 4.5 ml of antibody (4.5 mg/ml)) . The bulk drug of the monoclonal antibody APN311 is produced in the form of a concentrate for the preparation of drugs for intravenous infusion.

- 20 043975

Table 6

Composition of the final product APN311

The product's name chimeric mouse-human monoclonal antibody against GD2 (chl4.18/CHO; APN311) Content 4.25-4.75 mg/ml (exact content per 1 ml may vary slightly from batch to batch and is listed on each bottle) Buffer 20 mM histidine, 5% sucrose, 0.01% Tween 20, water for injection pH value 5.5-6.5 Filler No

Cooking instructions.

The antibody must be prepared under sterile conditions. An appropriate volume of ch14.18/CHO antibody (APN311) should be removed from the vial. It is recommended that the antibody solution be filtered (0.2 to 1.2 µm) before administration to the patient, either by using an inline filter during infusion (as some centers routinely do) or by filtering the solution through a particle filter (e.g. Filter Nr. MF1830, Impromediform, Germany). The volume of antibody is added to an infusion bag containing 0.9% NaCl and 1% human serum albumin (HSA), for example, 100 ml of 0.9% NaCl and 5 ml of 20% human serum albumin.

Calculation of the amount of ch14.18/CHO (APN311) for breeding.

The amount of ch14.18/CHO (APN311) to be administered is calculated as follows. Dosage: 10 mg/ ^m2 /day, days 8-17, as a 24-hour infusion.

Calculation example: If a patient has a body surface area (BSA) of 0.7, then 7 mg (10x0.7) per day is needed or 70 mg for 10 days of treatment (one cycle).

Example 2: Patients treated with APN311 without IL-2 (and without other cytokines), compared with data from Yu et al. 2010, NEJM (cited above).

The data underlying this example were obtained from the SIOP-Europe High Risk Neuroblastoma Study 1.5 (SIOPEN), EudraCT number 2006-001489-17. Patients with high-risk neuroblastoma after myeloablative therapy (MAT) and autologous stem cell transplantation (ASCR), as well as, depending on the circumstances, other therapies such as radiation, other chemotherapy, surgery, etc. received up to 5 cycles of 20 mg/m ² /day ch14.18/CHO administered as an 8-hour IV infusion for 5 consecutive days (days 22 to 26 of the treatment cycle) in 28-day cycles. This corresponds to a total ch14.18/CHO dose of 100 mg/ ^m2 /cycle. cis-RA was administered orally twice daily at a dose of 160 mg/m ² /day from days 1 to 14 of the treatment cycle (two equal doses per day, i.e. 2x80 mg/m ² ). One group did not receive IL-2 during the treatment cycle or for the entire duration of treatment. Patients in the other group received IL-2 at a dose of 6x106 IU/m ² /day from days 15 to 19 and from days 22 to 26 of the treatment cycle. Patients were treated for 6 treatment cycles, with treatment cycles 1-5 being as described above and treatment cycle 6 being a treatment period of isotretinoin alone (no antibody and no IL-2).

Event-free survival (EFS) and overall survival (OS) were analyzed. EFS and OS were also analyzed in the subgroup of patients who appeared to be fully responsive at the outset of antibody treatment (with no evidence of tumor at all evaluations performed). EFS and OS data in this subgroup were compared with EFS and OS data from the antibody, IL-2 and GM-CSF and isotretinoin (cis-RA) treatment regimen as described in Yu et al. (NEJM 2010, link above), which are included in the table. 7.

Patients in Yu et al. received ch14.18/SP2/0 at a dose of 25 mg/m ² /day for 4 consecutive days, and doses of ch14.18/SP2/0 were administered by intravenous infusion over 5.75 hours or 10 hours (can be extended to 20 hours with expected toxicity). IL-2 was administered every second cycle (eg, cycles 2 and 4) as a continuous IV infusion for 4 days (96 hours) at 3 mIU/m ² /day in the 1st week of the treatment cycle and 4.5 mIU/m ² /day in the 2nd week of the treatment cycle (i.e. 30 mIU/m ² /cycle). GM-CSF was administered subcutaneously at a daily dose of 250 μg/ ^m2 for 14 days (from day 0 to day 13). The total treatment time is 5 treatment cycles. See also table. 2, 3 and 4 regarding appropriate treatment regimens. The EFS and OS values in Yu et al. shown here in FIG. 2 and 3, for comparison, include only those fully responsive to the above treatment. Thus, only the subgroup fully responsive to the treatment of the invention was used for direct comparison.

Table 7

2-Year Relapse-Free Survival (EFS) and Overall Survival (OS) in Completely Receptive (CR) APN311-302 Compared to Yu et al. (NEJM 2010, cited above) percentage

2-year EFS in % 2-year OS in % CR subgroup at the start of randomization in the APN311-302 trial: APN311+cis-CA 70 81 NEJM 2010: chl448+IL2+GM-CSF+uHC-RA 66 86 NEJM 2010: cis-RA only 46 75

The results are also presented in Fig. 1, 2 and 3 (also in comparison with Yu et al.) and FIG. 4-15 (more on toxicity, i.e. observed adverse effects or side effects). According to these data, IL-2 does not contribute to the effectiveness of anti-GD2 antibody treatment. However, concomitant administration of IL-2 significantly increases side effects and toxicity. Treatment with AR#11+cis-IA in those who are fully susceptible from the very beginning of immunotherapy gives 2-year relapse-free survival and overall survival and overall survival curves similar to those in the ch14.18/SP2-0+IL2+GM-CSF+ group cis-RA in the NEJM 2010 study (Yu et al., link above).

This suggests that the addition of both IL2 and GM-CSF is not necessary to achieve the same level of effectiveness of the combination treatment as described in NEJM 2010.

EFS and OS values in APN311+cis-RA (fully responsive from the start of immunotherapy) are improved compared with EFS and OS in NEJM 2010 patients treated with cis-RA alone (2y-EFS: 70% vs 46%; 2y-OS : 81% vs. 75%; also see Table 7). This indicates the effectiveness of APN311 monotherapy.

Therefore, the overall risk-benefit assessment concluded that APN311+cis-RA, in those fully responsive from the outset of immunotherapy, may even be superior to the 4-component immunotherapy described in NEJM 2010, especially due to the significant reduction in side effects and reduction in treatment complexity due to the exclusion of IL2 and GM-CSF, and also due to similar clinical efficacy in terms of EFS and OS.

Example 3: CDC and WBT analysis in a patient with neuroblastoma during antibody treatment.

The patient with neuroblastoma received APN311 in a continuous infusion regimen, i.e. received the antibody as a continuous infusion over 24 hours a day for 10 consecutive days (from 1 to 10 days of the treatment cycle) at a dose of 10 mg/m ² /day for 6 consecutive cycles. Each treatment cycle lasted 35 days. During each treatment cycle and for the entire treatment period of these 6 consecutive cycles, the patient received only antibody treatment and did not receive any cytokines or retinoids.

Blood samples were collected from this patient at the indicated time points and analyzed by CDC and WBT methods as described in Examples 4 and 5. The results are presented in FIG. 16. From FIG. 16 shows that treatment with the antibody without the use of cytokines caused an increase in the level of tumor cell lysis. These levels of tumor cell lysis are comparable to those in patients treated with antibody and IL-2 (one representative patient is shown in FIG. 17). The patient shown in FIG. 17 received the same treatment as the patient shown in FIG. 16, but additionally received IL-2 subcutaneously at a dose of 6x106 IU/ ^m2 /day from days 1 to 5 and from days 8 to 12.

As can be seen, especially in FIG. 16B, the increased level of target cell lysis in the whole blood test (WBT) persists even during periods of time within the total treatment time when the patient did not receive an anti-GD2 antibody formulation, i.e. between periods of treatment with a drug containing an anti-GD2 antibody.

Example 4: CDC Assay Procedure.

The principle of the CDC method (complement dependent cytotoxicity).

The degree of cytotoxicity to tumor cells of normal human serum or plasma in the presence of APN301 or APN311 or patient serum or plasma after infusion of one of these antibodies on the GD2 antigen-positive neuroblastoma cancer cell line LAN-1 (target cells) is determined by the ⁵¹ Cr release assay. Target cells are incubated with Na2 ⁵¹ Cr(VI)O4, which penetrates the cell membrane and binds to cytoplasmic proteins in the form of reduced III-valent Cr, which no longer flows out of intact cells. When these cells are lysed after incubation with serum or plasma and antibodies or with patient serum or plasma, radioactivity is released into the supernatant depending on the lytic potential of the samples being tested.

In each individual experiment, spontaneous background lysis and complete lysis (maximum achievable cell lysis or maximum possible lysis of target cells) under the influence of a surfactant are determined. After subtracting the spontaneous lysis, the lysis caused by the test samples is calculated as a % of the total lysis.

Obtaining serum or plasma samples.

Whole blood samples were collected from healthy donors or patients using heparinized Vacutainer vials for plasma or clotting vials for serum. The vials were centrifuged at 2000xg for 20 min. Plasma or serum supernatants were used immediately for analysis or stored at -20°C (thawing and re-freezing were not allowed).

Labeling of target cells with ⁵¹ Cr.

LAN-1 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. The day before analysis, they were transferred to new flasks with fresh medium. The assay was performed in 96-well flat-bottom cell culture plates using 4 x 104 labeled cells per well with 800 nCi of ⁵¹ Cr activity per well. The required number of cells was taken from the culture flasks, the suspension was centrifuged and resuspended in 1 ml of PBS with 0.1% EDTA and 1% FCS. Added

- 22 043975 calculated the volume of ⁵¹ Cr solution and incubated the cells for 90 minutes at 37°C and 5% CO2 with careful rotation of the tubes.

The cell suspension was then washed twice with cell culture medium to remove radioactivity from outside the cells. This medium additionally contained 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. The sediment of labeled cells after the washing step was resuspended to the required concentration of 4x10 ⁵ cells/ml.

Analysis procedure.

To assess the cytolytic ability of antibodies, the following was pipetted:

µl of sample (antibody dilution);

100 µl of normal human serum or plasma diluted 1:4;

100 µl of ⁵¹ Cr-labeled cell suspension (4x10 ⁵ per ml).

To assess the cytolytic ability of patient plasma or serum, the following was pipetted:

µl of medium;

100 µl of patient’s plasma or serum diluted 1:4;

100 µl of ⁵¹ Cr-labeled cell suspension (4x10 ⁵ per ml).

CDC assay plates were incubated in a _CO2 incubator at 37°C, 5% CO2, for 4 hours, and for direct comparison with WBT for 20 hours.

In addition, an aliquot from each blood sample was preincubated with ganglidiomab, a mouse monoclonal antibody against ch14.18 (anti-idiotypic antibody), and then processed as described above. It specifically binds to the antibody present in the blood sample after the antibody is administered to the patient. In this case, the anti-idiotypic antibody inhibits the ability of this antibody to cause lysis of target tumor cells in combination with blood cells (WBT) and/or plasma components (CDC). Residual lysis can be defined as an effect not mediated by the antibody.

Supernatants were then collected from each well using a frame harvester with absorption cartridges and a suction press (Skatron). These cartridges, impregnated with cell supernatants, were transferred to the measurement vials of a gamma counter. In all samples, radioactivity, which is proportional to the release of ⁵¹ Cr after damage to labeled target cells, was measured and expressed in counts per minute (cpm). The results were calculated in % lysis minus the cpm values for spontaneous lysis in each sample and relative to the cpm values for the maximum achievable lysis with surfactant, which was taken as 100%:

|srsh sample - arsh with spontaneous lysis % lysis in the sample = x00.

SRS with maximum lysis - with spontaneous lysis

The CDC assay procedure described above was used for the results presented in FIG. 16A.

Example 5. WBT technique.

The principle of the WBT method.

The degree of cytotoxicity to tumor cells of normal human whole blood in the presence of APN301 or APN311 or patient whole blood after infusion of one of these antibodies on the GD2 antigen-positive neuroblastoma cancer cell line LAN-1 (target cells) is determined by the ⁵¹ Cr release assay.

Target cells are incubated with Na ₂ ⁵¹ Cr(VI)O ₄ , which penetrates the cell membrane and binds to cytoplasmic proteins in the form of reduced 111-valent Cr, which no longer leaks out of intact cells. When these cells are lysed after incubation with whole blood and antibodies or with whole blood from patients, radioactivity is released into the supernatant depending on the lytic potential of the samples being tested.

In each individual experiment, spontaneous background lysis and complete lysis (maximum achievable cell lysis or maximum possible lysis of target cells) under the influence of a surfactant are determined. After subtracting the spontaneous lysis, the lysis caused by the test samples is calculated as a % of the total lysis.

Obtaining blood samples.

Whole blood samples were collected from healthy donors or patients using heparinized Vacutainer vials.

Labeling of target cells with ⁵¹ Cr.

LAN-1 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. The day before analysis, they were transferred to new flasks with fresh medium. The assay was performed in 96-well flat-bottomed cell culture plates using 4 x 104 labeled cells per well with 800 nCi of ⁵¹ Cr activity per well. The required number of cells was taken from the culture flasks, the suspension was centrifuged and resuspended in 1 ml of PBS with 0.1% EDTA and 1% FCS. The calculated volume of ⁵¹ Cr solution was added and the cells were incubated for 90 min at 37°C and 5% CO ₂ with careful rotation of the tubes.

The cell suspension was then washed twice with cell culture medium to remove

- 23 043975 dioactivity outside the cells. This medium additionally contained 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. The sediment of labeled cells after the washing step was resuspended to the required concentration of 4x10 ⁵ cells/ml.

Analysis procedure.

To assess the cytolytic ability of antibodies, the following was pipetted:

µl of sample (antibody dilution);

100 µl of 1:2 diluted normal human whole blood;

100 µl of ⁵¹ Cr-labeled cell suspension (4x10 ⁵ per ml).

To assess the cytolytic ability of patients' whole blood, the following was pipetted: 50 μl of medium;

100 µl of patient’s blood diluted 1:2;

100 µl of ⁵¹ Cr labeled cell suspension (4x10 ⁵ per ml)

Assay plates were incubated in a _CO2 incubator at 37°C, 5% CO2, for 20 hours.

In addition, an aliquot from each blood sample was preincubated with ganglidiomab, a mouse monoclonal antibody against ch14.18 (anti-idiotypic antibody), and then processed as described above. It specifically binds to the antibody present in the blood sample after the antibody is administered to the patient. In this case, the anti-idiotypic antibody inhibits the ability of this antibody to cause lysis of target tumor cells in combination with blood cells (WBT) and/or plasma components (CDC). Residual lysis can be defined as an effect not mediated by the antibody.

Supernatants were then collected from each well using a frame harvester with absorption cartridges and a suction press (Skatron). These cartridges, impregnated with cell supernatants, were transferred to the measurement vials of a gamma counter. In all samples, radioactivity, which is proportional to the release of ⁵¹ Cr after damage to labeled target cells, was measured and expressed in counts per minute (cpm). The results were calculated in % lysis minus the cpm values for spontaneous lysis in each sample and relative to the cpm values for the maximum achievable lysis with surfactant, which was taken as 100%:

srsh sample - srsh with spontaneous lysis % lysis in the sample =*100.

SRS with maximum lysis - with spontaneous lysis

The WBT technique described above was used for the results presented in FIG. 16V.

The invention is further disclosed in the following embodiments, which can be easily combined with any of the claims.

1. A drug containing an anti-GD2 antibody for use in the treatment of GD2-positive cancer in a patient, wherein the drug containing an anti-GD2 antibody is administered to the patient without concurrent administration of IL-2, wherein one or more periods of administration of the antibody are preceded, followed and followed by /or followed by one or more periods of taking a retinoid.

2. An anti-GD2 antibody-containing formulation for use in the treatment of GD2-positive cancer in a patient, wherein the anti-GD2 antibody-containing formulation is administered to the patient without concurrent administration of IL-2, wherein one or more periods of antibody administration are preceded, followed, and followed by /or followed by one or more periods of retinoid dosing within the same treatment cycle.

3. An anti-GD2 antibody-containing preparation for use in treating GD2-positive cancer in a patient, wherein the anti-GD2 antibody-containing preparation is administered to the patient as a continuous infusion over one or more days without concurrent administration of IL-2.

4. An anti-GD2 antibody-containing preparation for use in the treatment of GD2-positive cancer in a patient, wherein the anti-GD2 antibody-containing preparation is administered to the patient as a continuous infusion over one or more days and over two or more treatment cycles without concurrent administration of IL-2.

5. A preparation containing a chimeric or humanized anti-GD2 antibody for use in the treatment of GD2-positive cancer in a patient, wherein the preparation containing the anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days without concurrent administration of IL-2.

6. An anti-GD2 antibody-containing preparation for use in treating GD2-positive cancer in a patient, wherein the anti-GD2 antibody-containing preparation is administered to the patient as a continuous infusion without concomitant administration of IL-2, and wherein the anti-GD2 antibody is not 14G2a antibody. .

7. A method of treating GD2-positive cancer in a patient, comprising administering an anti-GD2 antibody to the patient without simultaneous administration of IL-2, thereby treating the GD2-positive cancer.

8. A method of treating GD2-positive cancer by administering to a patient a drug containing an anti-GD2 antibody, wherein the patient does not simultaneously receive IL-2, wherein one or more periods of administration of the antibody are preceded, accompanied and/or followed by one or more periods of administration retinoid.

- 24 043975

9. A method of treating GD2-positive cancer by administering to a patient a drug containing an anti-GD2 antibody, wherein the patient does not simultaneously receive IL-2, and wherein one or more periods of administration of the antibody are preceded, followed and/or followed by one or more periods of administration taking a retinoid within the same treatment cycle.

10. A method of treating GD2-positive cancer by administering to a patient a drug containing an anti-GD2 antibody, wherein the drug containing an anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days without simultaneous administration of IL-2.

11. A method of treating GD2-positive cancer in a patient, wherein the drug containing an anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days and over two or more treatment cycles, and wherein the patient does not simultaneously receive IL-2 .

12. A method of treating GD2-positive cancer in a patient, wherein a drug containing a chimeric or humanized anti-GD2 antibody is administered to the patient as a continuous infusion over one or more days without concurrent administration of IL-2.

13. A method of treating GD2-positive cancer in a patient, wherein a drug containing an anti-GD2 antibody is administered to the patient as a continuous infusion, wherein the patient does not simultaneously receive IL-2, and the anti-GD2 antibody is not a 14G2a antibody.

14. The drug or method according to any of the previous embodiments, wherein the patient does not simultaneously receive GM-CSF.

15. The drug or method according to any of the previous embodiments, wherein the patient does not receive cytokines.

16. The drug or method according to any of the previous embodiments, wherein the patient does not receive cytokines within the same treatment cycle.

17. The drug or method according to any of the previous embodiments, wherein the patient does not receive cytokines within the same total treatment time.

18. The drug or method according to any of the previous embodiments, wherein the patient may have received IL-2, GM-CSF and/or one or more other cytokines in one or more previous treatment cycles and/or throughout treatment.

19. The formulation or method of any of the preceding embodiments, wherein the GD2-positive cancer is neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung cancer, desmoplastic small round cell fibroma, osteosarcoma, rhabdomyosarcoma, and/or other soft tissue sarcoma.

20. The drug or method according to any of the previous embodiments, wherein the GD2-positive cancer is neuroblastoma.

21. The drug or method according to any of the previous embodiments, wherein the patient has been diagnosed with high-risk neuroblastoma and/or stage 4 neuroblastoma.

22. The drug or method according to any of the previous embodiments, wherein the patient has been diagnosed with minimal residual disease.

23. The drug or method according to any of the previous embodiments, wherein the patient has been diagnosed with a relapsed or refractory disease.

24. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered as a continuous intravenous infusion over 24 hours per day.

25. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered in a daily dose of 1-15, 1-20, 1-25, 1-30, 1-35, 1-50 or 1- 60 mg/ ^m2 .

26. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered at a daily dose of 1-30, 1-35, 1-50 or 1-60 mg/m ² .

27. The drug or method according to any of the previous embodiments, wherein the drug containing an anti-GD2 antibody is administered at a dose of 10, 20, 25, 50, 60, 65, 68, 70, 75, 80, 100, 120, 150, 200 , 210, 250 or 300 mg/ ^m2 /cycle.

28. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered using a mini-pump.

29. The drug or method according to any of the previous embodiments, wherein the anti-GD2 antibody is represented by antibody 14.18.

30. The drug or method according to any of the previous embodiments, wherein the anti-GD2 antibody is encoded by the amino acid sequence of SEQ ID NO: 3 and/or 4 or fragments or homologues thereof with the same or similar qualitative activity.

31. The drug or method according to any of the previous embodiments, wherein the anti-GD2 antibody is ch14.18/CHO or ch14.18/SP2/0.

32. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is represented by APN311.

33. The drug or method according to any of the previous embodiments, wherein the anti-GD2 antibody is dinutuximab.

34. The drug or method according to any of the previous embodiments, wherein the drug containing

-

Claims (15)

antibody against GD2, administered at a dose of 7, 10, 15, 17.5, 20 or 25 mg/m ² /day. 35. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered for 4, 5, 10, 14, 15 or 21 consecutive days. 36. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is administered over 3, 4, 5, 6, 7, 8 or 9 or more treatment cycles. 37. The drug or method according to any of the previous embodiments, wherein the anti-GD2 antibody drug is APN311 and is administered at a dose of 10 mg/m ² /day for 10 consecutive days over 6 or more treatment cycles. 38. The drug or method according to any of the previous embodiments, wherein the drug containing the anti-GD2 antibody is represented by ch14.18/SP2/0 and is administered at a dose of 25, 20, 17.5 or 15 mg/m ² /day for 4 consecutive days for 5 or more treatment cycles. 39. The drug or method according to any of the previous embodiments, wherein the period of administration of the drug containing the anti-GD2 antibody may be followed by a period of administration of isotretinoin or other retinoid. 40. The drug or method according to any of the previous embodiments, wherein the administration of the drug containing the anti-GD2 antibody is accompanied by the administration of morphine and/or one or more other analgesics. 41. The drug or method according to any of the previous embodiments, wherein the administration of the drug containing the anti-GD2 antibody is accompanied by the administration of a reduced dose of morphine and/or one or more other analgesics. 42. The drug or method according to any of the previous embodiments, wherein the administration of the drug containing the anti-GD2 antibody is not accompanied by the administration of morphine and/or one or more other analgesics. CLAIM 1. A method of treating GD2-positive cancer in a patient, comprising administering to the patient a drug containing a ch14.18 antibody, without simultaneous administration of IL-2, within the same treatment cycle and/or within the same total treatment time, wherein the ch14.18 antibody administered to the patient at a dose of 1 to 25 mg/m ² /day and from 50 to 150 mg/m ² /cycle as a continuous intravenous infusion for 24 hours per day, while treating GD2-positive cancer. 2. The method of claim 1, further comprising administering to the patient a retinoid within the same treatment cycle before, during and/or after administration of the ch14.18 antibody. 3. The method according to claim 1 or 2, wherein the anti-GD2 antibody is ch14.18/CHO or ch14.18/SP2/0. 4. The method according to any one of claims 1 to 3, wherein the anti-GD2 antibody has a light chain amino acid sequence of SEQ ID NO: 3 and a heavy chain amino acid sequence of SEQ ID NO: 4. 5. Method according to any one of claims 1-4, wherein the drug is administered to the patient at a dose of 50, 60, 65, 68, 70, 75, 80, 100, 120 or 150 mg/ ^m2 /cycle. 6. Method according to any one of claims 1-5, wherein the drug is administered to the patient in a daily dose of 1-15 or 1-20 mg/ ^m2 . 7. Method according to any one of claims 1-6, wherein the drug is administered to the patient in a daily dose of 7, 7.5, 8, 9, 10, 12, 13, 14, 15, 16, 17, 17.5, 18, 19, 20 or 25 mg/ ^m2 . 8. The method according to any one of claims 1 to 7, wherein the drug is administered to the patient at a dose of 10 mg/m ² /day for 10 consecutive days or at a dose of 15, 17.5, 20 or 25 mg/m ² /day for 4 consecutive days. 9. The method according to any one of claims 1 to 8, wherein the ch14.18 antibody is administered to the patient over 2 or more treatment cycles. 10. The method according to any one of claims 1 to 9, wherein the patient does not simultaneously receive GM-CSF within the same treatment cycle and/or within the same total treatment time. 11. The method according to any one of claims 1 to 10, wherein the patient does not simultaneously receive a cytokine within the same treatment cycle and/or within the same total treatment time. 12. The method according to any one of claims 1 to 11, wherein the patient does not receive IL-2, and/or GM-CSF, and/or a cytokine within the same treatment cycle and/or within the same general treatment time. 13. The method according to any one of claims 1 to 12, wherein the GD2-positive cancer is neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung cancer, desmoplastic small round cell fibroma, osteosarcoma, rhabdomyosarcoma and/or other soft tissue sarcoma. 14. The method according to any one of claims 1 to 13, wherein the patient suffers from primary refractory or recurrent high-risk neuroblastoma or minimal residual disease from high-risk neuroblastoma. 15. The method according to any one of claims 1 to 14, wherein the daily dose of morphine administered during one or more days of administration of the drug containing the ch14.18 antibody in accordance with the invention, -