RU2733430C1 - Creation of conjugates of gd2-specific antibodies and fragments of gd2-specific antibodies with preparations - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to biochemistry, particularly to an immunoconjugate for treating GD2-positive oncological diseases. Also disclosed is a composition containing said immunoconjugate. Disclosed is a method of treating a GD2-specific tumor disease using said immunoconjugate.EFFECT: invention provides effective treatment of diseases associated with GD2.9 cl, 8 dwg, 6 ex

Description

Technology area

The invention relates to biotechnology and can be used to obtain constructs based on antibodies to tumor-associated ganglioside GD2. These antibodies and their fragments can find application in medicine, immunology, and biotechnology for the preparation of therapeutic drugs for the treatment of GD2-positive oncological diseases.

State of the art

The global market for antibody-drug conjugates (ADCs) is projected to have significant prospects, with a compound annual growth rate (CAGR) to 2023 of 19%. The ADCs market is considered quite open, and there is a demand for developments aimed at creating new, more efficient and safer molecules, as well as optimizing the methods for their creation. There are currently only 5 FDA approved ADCs on the market. About 80 ADCs are in approximately 600 clinical trials for the treatment of both hematologic and solid tumors. The great interest in this technology is due to the fact that ADCs can combine the best therapeutic qualities of their constituent components, namely, the selectivity of the action of monoclonal antibodies will be combined with a high cytotoxic effect of chemotherapy drugs.

The strategy for creating ADCs is to reduce the systemic toxicity characteristic of chemotherapy drugs, as well as to enhance the effector properties of therapeutic antibodies. For the activity of such conjugates, in addition to internalization into the cell, the release of the drug is also necessary, since usually the construct itself does not have cytotoxic activity. The ratio of chemotherapy to vector molecule is not large and is 3-10 molecules per antibody or antibody fragment, since more drug can lead to a loss of binding capacity of the vector molecule (Allen TM. Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer. 2002 Oct; 2 (10): 750-63).

Monoclonal antibodies form the backbone of ADCs and recognize antigens present on cancer cells. Such an antigen must be internalized in order for the drug bound to the antibody to be transported into the cell, where this cytotoxic agent performs its antitumor function (Damelin M, et al., Evolving strategies for target selection for antibody-drug conjugates. Pharm Res 2015; 32 : 3494-507). It should be noted that antibodies themselves are capable of performing an antitumor function, for example, by reducing signals that stimulate tumor progression, or by directly inducing cell death of tumor cells (Doronin II, et al., Ganglioside GD2 in reception and transduction of cell death signal in tumor cells. BMC Cancer. 2014 Apr 28; 14: 295), as well as through Fc-mediated effector mechanisms. However, the presence of anti-tumor activity of antibodies is not a key requirement when creating ADCs.

There are only a few papers in the scientific literature on the use of antibody fragments to generate ADCs. So in the work of Kim et al. Conjugates of anti-CD30 diabodi with anti-tubulin drugs, monomethyl auristatin E or F were obtained. The drugs were conjugated at a ratio of 4 molecules per 1 molecule of diabodi via a dipeptide linker cleaved by proteases. These ADCs had high in vitro cytotoxicity and significant anti-tumor response in the xenograft model at well tolerated concentrations. At the same time, the antitumor response of diabodi ADCs was slightly inferior to the corresponding ADCs based on full-length antibodies (Kim, KM, et al. Mol. Cancer Ther. 2008. 7, 2486-2497).

Ganglioside GD2 is a marker of many tumors, including neuroblastoma, glioma, melanoma, various sarcomas, and small cell lung cancer. In normal cells, this molecule is practically not present, with the exception of minor expression on brain cells, in peripheral nerves and skin melanocytes. During tumor transformation, the expression of ganglioside GD2 increases by orders of magnitude, reaching 10 million molecules per cell, which makes it a promising target for anticancer therapy (Ahmed M., and Cheung NK Engineering anti-GD2 monoclonal antibodies for cancer immunotherapy. FEBS Lett, 2014.588 ( 2): 288-97). The most promising approach for using the onco-associated GD2 ganglioside in anticancer therapy is immunotherapy using monoclonal antibodies. One of the main advantages of cancer immunotherapy is that immune effector cells recognize and destroy tumor cells that are inaccessible to traditional therapies with exceptional specificity, while generating long-term immune surveillance against cancer cells. In the mid-1980s, preclinical studies showed significant antitumor effects of murine GD2-specific antibodies in various animal models of cancer (Katano M, Irie RF. Immunol Lett. 1984; 8 (4): 169-74). However, so far only one drug, Unituxin (based on the chimeric GD2-specific antibody Dinutuximab) has been approved for clinical use. Unituxin has significant efficacy and a 20% increase in 3-year survival in high-risk neuroblastoma patients in combination therapy compared with chemotherapy alone.

However, the use of Unituxin has a number of limitations, including insufficient effectiveness and side effects. The limited effectiveness of the action of chimeric GD2-specific antibodies is largely due to the low activity of immune effector cells inside solid tumors, as well as the large size of antibodies that poorly penetrate dense neoplasms. To a large extent, these limitations can be overcome by obtaining immunoconjugates with drugs, as well as by using antibody fragments (Kholodenko RV, et al. Curr Med Chem. 2019; 26 (3): 396-426).

The aim of this study was to create new conjugates of antibodies (or antibody fragments) with drugs effective in the elimination of GD2-positive tumor cells. This invention expands the range of candidate molecules available for the treatment of GD2-positive cancers.

The essence of the invention

The objective of the present invention is to create a therapeutic agent based on antibodies to GD2 and to develop a new effective and safe method for treating patients with cancer, characterized by the presence of GD2 ganglioside on the surface of tumor cells. It is also an object of the present invention to expand the arsenal of agents for suppressing proliferation and inducing death of GD2-positive tumor cells. For this purpose, purified chimeric GD2-specific antibodies and scFv-fragments of antibodies were obtained for subsequent conjugation of cytotoxic drugs belonging to the class of dolastins and maytansinoids through cleavable and non-cleavable linkers. One of the problems solved in the present invention was the development of methods for covalent attachment of cytotoxic drugs carrying a linker to GD2-specific antibodies and scFv fragments, as well as purification of the resulting conjugates. The analysis of the cytotoxic effect of the obtained conjugates of GD2-specific antibodies and antibody fragments with cytotoxic drugs was carried out on GD2-positive and GD2-negative tumor lines. The antitumor effects generated by ADCs were evaluated in a relevant syngeneic mouse cancer model.

These problems were solved by creating a new therapeutic agent for the treatment of GD2-specific tumor diseases, which is an immunoconjugate that specifically binds the GD2 ganglioside and has the general formula: A- (L-C) n, where (a) A is an antibody, an antibody fragment or a multimeric an antibody fragment that includes from one to four antigen-binding regions having a heavy chain (or part of it) containing H-CDR1 with the sequence SEQ ID NO: 1, H-CDR2 with the sequence SEQ ID NO: 2, and H-CDR3 with SEQ ID NO: 3, and a light chain (or part thereof) containing L-CDR1 with the sequence SEQ ID NO: 4, L-CDR2 with the sequence SEQ ID NO: 5, and L-CDR3 with SEQ ID NO: 6; (b) L is a linker; (c) C is a cytotoxic or cytostatic agent; and (d) n ranges from 1 to 8.

In some embodiments, the immunoconjugate is characterized in that the heavy chain contains a variable domain with a sequence homologous to at least 95% of SEQ ID NO: 7. In some embodiments, the immunoconjugate is characterized in that the light chain contains a variable domain with a sequence homologous to at least 95% of SEQ ID NO: 8.

In some embodiments, the immunoconjugate may contain full length anti-GD2 antibodies.

In some embodiments, the immunoconjugate is characterized in that the cytotoxic or cytostatic agent is selected from the group of monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, maytansinoid DM4, or maytansinoid DM1.

In some embodiments, the immunoconjugate is characterized in that n is 2, 4, 6, or 8.

In some embodiments, the immunoconjugate is characterized in that A is a multivalent construct containing two, three, or four antigen-binding antibody fragments linked by covalent conjugation to a polyethylene glycol molecule.

This problem is also solved by creating a pharmaceutical composition for the treatment of a GD2-specific tumor disease, containing the above-described immunoconjugate in a therapeutically effective amount, as well as a pharmaceutically acceptable carrier, diluent and / or filler. In some embodiments, the GD2-specific tumor disease is neuroblastoma or other GD2-positive tumors (glioma, small cell lung cancer, breast cancer, sarcoma).

This problem is also achieved by providing a method for treating a GD2-specific tumor disease in a subject in need thereof, comprising administering to the subject an effective amount of the above-described composition.

The technical result achieved by the implementation of the invention is the creation of a new, effective version of a therapeutic agent based on antibodies to GD2 ganglioside for the treatment of a GD2-specific tumor disease. The resulting therapeutic agent has a number of properties necessary for its effective and safe use, including the agent has increased cytotoxicity in vitro and in vivo in relation to cells containing GD2 ganglioside on the surface. Variants of the agent containing antigen-binding fragments of antibodies, but not full-length antibodies, are characterized by the absence of unwanted effector functions, as well as by a reduced size, which ensures better penetration of the agent into tumor tissues. The multivalence of the fragments with the help of the PEG molecule gives them stability and an increased residence time in the bloodstream.

Brief description of figures

FIG. 1. A - 12% PAGE electrophoresis of chimeric antibodies 14.18 (ch14.18) under reducing conditions. Lane 1 - markers, lane 2 - ch14.18

B - 12% PAGE electrophoresis of scFv fragments 14.18 under reducing conditions. Lane 1 - markers, lane 2 - scFv 14.18

B - Western blot of scFv fragments 14.18. Incubation with anti-FLAG antibodies (1: 6000). Visualization using 1-Step Ultra TMB-Blotting Solution.

FIG. 2. Structure of a conjugate of a GD2-specific antibody with monomethyl auristatin E.

FIG. 3. A - 12% PAGE electrophoresis in reducing conditions of chimeric antibodies 14.18, scFv fragments and conjugates of antibodies with a drug based on them. 1 - ch14.18-MMAF, 2 - ch14.18, 3 - scFv 14.18, 4 - scFv-MMAE.

B - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the substrate. Added serial dilutions of ch14.18 antibodies and ch14.18-MMAE drug conjugates (ADCs) in two antibody: chemotherapy molar ratios. After incubation with HRP-labeled anti-human antibodies (1: 6000), the reaction was developed with Ultra TMB-ELISA Substrate Solution.

B - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the support. Added serial dilutions of antibodies ch14.18 and ADCs (ch14.18-MMAE and ch14.18-MMAF). After incubation with HRP-labeled anti-human antibodies (1: 6000), the reaction was developed with a TMB substrate (substrate-chromogenic mixture TMB, Immunotech).

D - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the support. Added serial dilutions of scFv antibody fragments. After incubation with HRP-labeled anti-FLAG antibodies (1: 6000), the reaction was developed with Ultra TMB-ELISA Substrate Solution.

FIG. 4. A. Structure of the conjugate of the GD2-specific antibody with DM1.

B - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the substrate. Added serial dilutions of antibodies ch14.18 and ADCs ch14.18-DM1. After incubation with HRP-labeled anti-human antibodies (1: 6000), the reaction was developed with Ultra TMB-ELISA Substrate Solution.

B - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the support. Added serial dilutions of scFv 14.18 and ADCs scFv 14.18-DM1. After incubation with HRP-labeled anti-human antibodies (1: 6000), the reaction was developed with Ultra TMB-ELISA Substrate Solution.

D - direct enzyme-linked immunosorbent assay, GD2 ganglioside is sorbed on the support. Added serial dilutions of tetra-scFv 14.18 and ADCs tetra-scFv14.18-DM1. After incubation with HRP-labeled anti-human antibodies (1: 6000), the reaction was developed with Ultra TMB-ELISA Substrate Solution.

FIG. 5. Flow cytometry. Staining of cells of lines IMR-32, EL-4 and NGP-127 with GD2-binding molecules. Control - intact cells.

In the MTT test on selected GD2-positive and GD2-negative cell lines, the cytotoxic effects of the resulting ADCs were tested.

FIG. 6. Evaluation of cell death by ADCs carrying MMAE or MMAF.

A - MTT test of EL-4, IMR-32 and NGP-127 cells incubated with сh14.18-MMAE for 72 h. B - MTT-test of EL-4, IMR-32 and NGP-127 cells incubated with сh14. 18-ММАF for 72 h. C - MTT test of EL-4 cells incubated with сh14.18-ММАE and сh14.18-ММАF for 72 hours. D - MTT test of EL-4 cells incubated with scFv 14.18-ММАE and scFv 14.18-MMAF for 72 hours.

FIG. 7. Evaluation of cell death by ADCs carrying DM1.

A - MTT test of EL-4, IMR-32 and NGP-127 cells incubated with сh14.18-DM1 for 72 h. B - MTT test of EL-4, IMR-32 and NGP-127 cells incubated with scFv 14.18 -DM1 for 72 h. C - MTT test of EL-4, IMR-32 and NGP-127 cells incubated with di-scFv 14.18-DM1 for 72 h. D - MTT test of EL-4, IMR-32 cells and NGP-127 incubated with tetra-scFv 14.18-DM1 for 72 hours.

FIG. 8. Evaluation of the antitumor effects of ADCs in a murine syngeneic GD2-positive cancer model. After tumor development (200-250 mm3), mice were injected intravenously with antibodies ch14.18, ADCs ch14.18-MMAF, or saline 4 times with an interval of 4 days.

Detailed disclosure of the invention

Definitions and terms

For a better understanding of the present invention, below are some of the terms used in the present description of the invention.

In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things". These terms are not intended to be construed as “consists only of”.

By "subject" is meant a human or other mammal.

Ganglioside GD2 in the present description refers to a disialoganglioside expressed on the surface of tumor cells of neuroectodermal origin with the following IUPAC formula: (2R, 4R, 5S, 6S) -2- [3 - [(2S, 3S, 4R, 6S) -6 - [( 2S, 3R, 4R, 5S, 6R) -5 - [(2S, 3R, 4R, 5R, 6R) -3-acetamido-4,5-dihydroxy-6- (hydroxymethyl) oxan-2-yl] oxy-2 - [(2R, 3S, 4R, 5R, 6R) -4,5-dihydroxy-2- (hydroxymethyl) -6 - [(E) -3-hydroxy-2- (octadecanoylamino) octadec-4-enoxy] oxan- 3-yl] oxy-3-hydroxy-6- (hydroxymethyl) oxan-4-yl] oxy-3-amino-6-carboxy-4-hydroxyoxan-2-yl] -2,3-dihydroxypropoxy] -5-amino -4-hydroxy-6- (1,2,3-trihydroxypropyl) oxane-2-carboxylic acid.

The term "antigen-binding, or functional, antibody fragment" as used herein means one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, GD2 ganglioside). Examples of antigen binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of one arm of an antibody; (iv) two Fv domains, VL and VH, joined by a synthetic linker into a single protein chain, in which the VL and VH regions are paired to form a monovalent construct (known as single chain Fv fragment (scFv); see, for example, Bird et al., Science, 1988, 242: 423-426). These antibody fragments are prepared by conventional methods known to those skilled in the art. In preferred embodiments of the present invention, antigen-binding antibody fragments lack or have reduced effector functions; in particular, antigen-binding antibody fragments may not contain an Fc region.

Some multivalent constructs of the present invention may contain from two to more antigen-binding antibody fragments, each of which is capable of specifically binding to GD2 ganglioside; thus, contain two or more antigen-binding sites. Antigen-binding fragments of antibodies in a multivalent construct can include variable regions, as well as, sometimes, constant regions isolated from immunoglobulin sequences that specifically bind GD2. Preferably, but not necessarily limited, the antigen binding fragments in the multivalent construct of the present invention lack or have reduced effector functions. Multivalence, i.e. the ability to bind several antigen molecules at once provides the constructs of the present invention with high avidity (stability of interaction between the construct and a cell expressing antigens on the surface), which in turn provides improved antigen-binding properties of the construct.

Used antigennegative antibody fragments of the present invention can be "chimeric" or "humanized" antibody fragments. Chimeric fragments mean antibody fragments that include heavy and / or light chain variable region sequences from one species and constant region sequences from another species. Humanized fragments means antibody fragments that include sequences of human antibody fragments in which some CDR residues and possibly some framework region (FR) residues have been replaced by residues from similar sites from rodent antibodies.

In some embodiments, antigen-binding antibody fragments of the invention comprise heavy and / or light chain regions including amino acid sequences homologous to amino acid sequences of preferred antibody fragments, wherein said antigen-binding antibody fragments retain the desired antigen-binding properties of the invention (binding to GD2 ganglioside and induction of cellular death). Homologous antibody fragments can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the corresponding nucleic acid molecules, followed by testing the encoded modified antibody fragment to maintain its function in accordance with the functional assays described herein.

As used herein, the term "percent homology of two sequences" is equivalent to the term "percent identity of two sequences." The percentage of identity of two sequences is determined by the number of positions of identical amino acids in the two sequences, taking into account the number of gaps and the length of each gap that must be entered for optimal alignment of the two sequences. The percentage of identity is equal to the number of identical amino acids at given positions, taking into account the alignment of the sequences, divided by the total number of positions and multiplied by 100. The percentage of identity of two amino acid sequences can be determined using the NCBI Protein BLAST program (https: //blast.ncbi.nlm.nih .gov /).

The term "pharmaceutically acceptable carrier, diluent and / or excipient" refers to such carriers, diluents and / or excipients that, being inactive ingredients, within the scope of the medical opinion, are suitable for use in contact with human and animal tissues without undue toxicity, irritation , allergic reaction, etc. and meet a reasonable balance of benefits and risks. "Inactive ingredients" are included in a drug or vaccine preparation to improve its solubility and / or stability, as well as to improve pharmacokinetics and more efficient delivery to specific organs or tissues. Inactive ingredients include many substances known to those skilled in the pharmaceutical arts such as pH or osmotic pressure control agents, antibacterial agents, antioxidants, surfactants (e.g. polysorbate 20 or 80), cryostabilizers, preservatives, solvents, thickeners, fillers , carriers (micro- or nano-particles) and other substances.

The term "therapeutically effective amount" means an amount of a pharmaceutical composition that, when ingested in a subject, will effectively induce the elimination of tumor cells expressing GD2 ganglioside. When used in combination therapy, the term "effective amount" refers to a combination of amounts of active ingredients, the administration of which leads to a preventive or therapeutic effect when taken sequentially or simultaneously. The exact amount required may vary from subject to subject depending on the mammalian species, the age and general condition of the patient, the severity of the disease, the route of administration of the drug, the combination of treatment with other drugs, and the like. The pharmaceutical composition described in the present invention can be administered to a patient in any amount and by any route of administration effective for the treatment and / or prevention of cancer.

Two-thirds of ADCs immunoconjugates in clinical trials carry two types of antimitotic compounds as cytotoxic agents: auristatins and maytansinoids (Beck, A. & Reichert, J. M. mAbs 2014.6, 15-17). This fact indicates the difficulty in the selection of cytotoxic molecules that would meet a number of requirements, often contradicting each other. These requirements include a high level of cytotoxic potential, relative hydrophilicity, lack of susceptibility to MDR1-mediated efflux, which is the main mechanism of resistance for ADCs (Salomon, P. L. & Singh, R. 2015. Mol. Pharm. 12, 1752-1761). The most commonly used cytotoxic agents are anti-microtubule compounds or DNA damaging compounds (Beck A, et al. Nat Rev Drug Discov 2017; 16: 315-37). The first group includes analogs of dolastatin 10-based auristatin (Senter PD, Sievers EL. Nat Biotechnol 2012; 30: 631-7.), Maytansinoids (Amiri-Kordestani L, et al. Clin Cancer Res 2014; 20: 4436 -41) and tubulysins (Li JY, et al. Cancer Cell 2016; 29: 117-29.). The second group includes calicheamicin analogs (Appelbaum FR, Bernstein ID. Blood 2017; 130: 2373-76.) And duocarmycin analogs (van der Lee MM, et al. Mol Cancer Ther 2015; 14: 692-703.). Other drugs are also used in the clinical development phase of ADCs, such as topoisomerase inhibitors (camptothecin analogs) (Nakada T., et al. Bioorg Med Chem Lett 2016; 26: 1542-45.) And DNA alkylators (pyrrolbenzodiazepines) (Mantaj J, et al. Angew Chem Int Ed Engl 2017; 56: 462-88.), as well as inhibitors of RNA polymerase II (e.g. α-amanitin) (Liu Y, et al. Nature 2015; 520: 697-701.). These agents were selected for their high cytotoxicity, such that they are capable of destroying tumor cells at the intracellular concentrations achieved after the internalization of ADCs. These agents are preferred for use in the present invention.

The linker used in the present invention to bind a cytotoxic agent to an antibody must be highly stable in circulation, since unstable binding can lead to the release of the cytotoxic agent still in the bloodstream, resulting in systemic toxicity and decreased efficacy (Thomas A, et al., Lancet Oncol 2016; 17: e254-e62.). However, it must provide effective drug release following internalization of ADCs. Currently, the clinic uses two main classes of linkers: cleavable and non-cleavable. Cleavable linkers can be processed chemically or enzymatically. Three different types of release mechanisms have been developed: (1) acid-sensitive linkers such as hydrazone linkers, which are cleaved in lysosomes as a result of low pH (Nolting B. Methods Mol Biol 2013; 1045: 71-100.); (2) linkers sensitive to lysosomal proteases such as valine-alanine and valine-citrulline peptide linkers that are cleaved by lysosomal enzymes (Dubowchik GM, et al. Bioconjug Chem 2002; 13: 855-69.); and (3) redox-sensitive linkers, such as disulfide bond linkers, which are reduced in the cytoplasm (Erickson HK, et al. Bioconjug Chem 2010; 21: 84-92). Non-cleavable linkers are highly stable in circulation and intracellularly, thus release of the cytotoxic agent is dependent on complete proteolytic cleavage of the antibody in lysosomes after ADCs are internalized. The active catabolite includes a cytotoxic agent attached to a linker still attached to an amino acid residue of an antibody (Erickson HK, et al. Cancer Res 2006; 66: 4426-33.). Examples of non-cleavable linkers are the thioether linker used in trastuzumab emtansine (Lewis Phillips GD, et al. Cancer Res 2008; 68: 9280-90.) Or maleimidocaproic acid linked to monomethylauristatin F (mc-MMAF) used in some in clinical trials, such as depatuxizumab mafodotin (Phillips AC, et al. Mol Cancer Ther 2016; 15: 661-9).

As one of the approaches in the present invention, it is proposed to use GD2-binding fragments of antibodies without the Fc-domain for the elimination of GD2-positive tumor cells. Previously, the authors showed that Fab-fragments of GD2-specific antibodies retain the ability to induce cell death in tumor cells (Doronin II et al., Bull Exp Biol Med , 2013, Mar; 154 (5): 658-63), despite the absence of Fc-associated effector functions. To increase the duration of the presence of antibody fragments in the bloodstream, the pegylation procedure was used - covalent conjugation with a polyethylene glycol (PEG) molecule. This method has been used successfully for other protein molecules on numerous occasions. This procedure actually increased the duration of the presence of GD2-binding antibody fragments in the bloodstream, but the efficiency of inducing cell death of tumor cells was not always sufficient. Unexpectedly, it has been found that the attachment of two or more GD2-binding antibody fragments to one PEG molecule increases the cytotoxic effects of GD2-binding antibody fragments to tumor cells [Kholodenko IV et al., Molecules 2019, 24 (21), 3835]. Such a multivalent construct also has a sufficiently long lifetime in the bloodstream, and, potentially, reduced side effects on nerve endings compared to standard antibodies. The point is that such a construct will not induce complement-dependent cytotoxicity due to the absence of the Fc region; at the same time, Fc-independent cytotoxicity strongly depends on the concentration of GD2 on the cell surface; therefore, the cytotoxic effect of the multivalent construct will be more specific for tumor cells. The presence of a PEG molecule in the multivalent construct improves the pharmacokinetic characteristics of the molecule by reducing hepatic clearance and decreasing absorption by the reticuloendothelial system; in addition, pegylation significantly increases the EPR-effect (enhanced permeation and retention effect) - the property of the drug to be predominantly localized in the tumor when administered intravenously. The drug accumulated as a result of passive targeted delivery in the tumor growth focus will actively and selectively interact only with tumor cells overexpressing the GD2 ganglioside. Thus, the described multimerization using a PEG molecule enhances both the pharmacokinetic and pharmacodynamic properties of the anti-GD2 antibody fragments.

The following examples are provided for the purpose of revealing the characteristics of the present invention and should not be construed as limiting the scope of the invention in any way.

Methods for obtaining fragments of GD2-specific antibodies used in the present invention have been described in RF patent No. 2663104, the contents of which are incorporated herein. Also, in the patent of the Russian Federation No. 2663104 data were presented confirming the improvement of the pharmacokinetic and pharmacodynamic properties of therapeutic agents containing multivalent constructs of antibody fragments.

Example 1. Materials and methods for the experiments described in this invention.

Cell cultivation.

For the cultivation of eukaryotic cells, the following media were used: complete medium (PS) based on one of the media, depending on the cell type: RPMI-1640 / RPMI-1640 Hybri-Max / DMEM / EMEM (pH 7.4) containing 10% FBS ( inactivated for 30 min at 56 ° C), 2 mM L-glutamine with the addition of penicillin / streptomycin.

Obtaining a conditioned medium of CHO cells producing GD2-specific antibodies 14.18, as well as scFv fragments of these antibodies. Cells were grown to concentrations critical for survival. The conditioned media from the vials were transferred into 50 ml test tubes and centrifuged at 300 g for 10 min. The supernatant was collected and transferred into new tubes, which were centrifuged for 15 min at 1000 g to remove cell debris. Before the isolation of antibodies and antibody fragments, the supernatant was additionally filtered through a filter with 0.4 μm pores.

Isolation of antibodies and scFv antibody fragments.

To isolate antibodies from the supernatant, a Protein A agarose column from the MAbTrap Kit Protein A reagent kit (GE Healthcare, USA) was used. After washing and activating the column with binding buffer, conditioned medium was applied to it. The column was washed to remove unbound proteins, then antibodies were eluted. The purified antibodies were collected and two successive ion exchange chromatographies were performed.

To isolate and purify scFv fragments from the conditioned medium, a Protein L agarose column from the HiTrap Protein L reagent kit (GE Healthcare, USA) was used. After washing and activating the column with binding buffer, the supernatant containing the scFv fragments was loaded onto it. The column was washed to remove unbound proteins, elution was performed with a buffer with pH 3.0, and then the pH of the solution was brought to neutral values with TRIS buffer. scFv fragments 14.18 were transferred into phosphate buffer (PBS) pH 7.2 on 10 kDa Amicon Ultra filters and sterilized by filtration through a 0.22 μm membrane. Protein concentration was determined spectrophotometrically at a wavelength of 280 nm on a BioDrop μLITE instrument (BioDrop, UK).

Multimerization of scFv fragments of GD2-specific antibodies through pegylation.

To enhance the therapeutic potential of fragments of GD2-specific antibodies and improve their pharmacokinetic properties, the authors used site-directed pegylation, the purpose of which was not only to increase the circulation time of fragments in the blood, but also to enhance cytotoxic properties by obtaining stable multimers of fragments of GD2-specific antibodies. To obtain di-, tri-, and tetramers of antibody fragments, we used standard reagents PEG-maleimide-2 and PEG-maleimide-4 (JenKem Technology USA, USA https://www.jenkemusa.com). The PEG derivatives used make it possible to attach several (from 1 to 4) fragment molecules to one PEG molecule.

To carry out the pegylation reaction of scFv fragments of monoclonal antibodies to a solution of fragments (2.5 mg / ml) in pegylation buffer (20 mM phosphate buffer, supplemented with 50 mM NaCl, 10 mM EDTA, pH 7.5), a stock solution of 15 mM TCEP was added until the final concentration of TCEP 0.1 mM. Incubation was carried out at room temperature for 90 min with gentle stirring. The reducing agent was removed by centrifugation through Amicon Ultra-4 filters (maximum throughput 10 kDa), or using Zeba Desalting Columns (TermoFisher, USA) for salting out. Then, the corresponding maleimide-PEG solutions were added to the solution of antibody fragments, in various ratios. Incubation lasted 16-18 hours at room temperature with gentle stirring on a shaker. The efficiency of pegylation was assessed using PAGE electrophoresis. After selecting the optimal conditions for carrying out pegylation, the yield of the reactions was at least 90%.

To separate the obtained pegylated fragments and obtain pure fractions containing individual mono- and multimers of antibody fragments, chromatographic purification was performed using anion exchange chromatography or gel filtration.

In one of the variants, cation exchange chromatography was performed on a TSK-GEL SP-5PW column using a Beckman System gold HPLC system to separate the obtained products of the pegylation reactions of antibody fragments, mobile phase: elution buffer (25 mM CH ₃ COOH, pH 4.0), linear gradient 0 mM - 250 mM NaCl (for 90 min at room temperature, flow rate 1 ml / min).

Alternatively, gel filtration (size exclusion chromatography) was used to separate the obtained products of pegylation reactions - a Superdex 200 10/30 GL column and PBS eluent passed through a 0.22 μm filter. Flow rate 0.6 ml / min.

Polyacrylamide gel electrophoresis of proteins and Western blot.

Electrophoresis was carried out in 12% acrylamide gel according to the Lemmli method. The gel was stained with Coomassie R250 dye. After electrophoresis, proteins from the gel were transferred onto a nitrocellulose membrane by the method of semi-dry transfer using a V10-SDB chamber (Biostep, Germany). Then, the sites of nonspecific binding of the membrane were blocked by incubation in a 5% solution of skimmed milk powder (Roth, Germany) for an hour with gentle rocking. After three washes in PBS containing 0.1% Tween 20, incubation with HRP-labeled anti-FLAG-specific antibodies 1: 6000 was carried out for an hour. The reaction of visualization of specific proteins on the membrane was performed using a 1-Step Ultra TMB-Blotting Solution (Thermo Scientific, USA) according to the manufacturer's method. Gels and membranes were analyzed on a Gel Doc EZ Imager and processed using the Image Lab software (Bio-Rad).

Preparation of conjugates of antibodies / antibody fragments with cytotoxic drugs MMAE / MMAF.

To obtain immunoconjugates ADCs used chimeric GD2-specific antibodies 14.18 or scFv fragments 14.18 (described in RF patent No. 2663104) and conjugate mc-vc-PAB-MMAE (or mc-vc-PAB-MMAF). This conjugate consists of a maleimidocaproyl group for binding to a thiol group of antibodies, a valine-citrulline dipeptide as a cleavable linker sensitive to lysosomal enzymes and cytotoxic substances monomethyl auristatin E or F. proteins (2.5 mg / ml) in a recovery buffer (20 mM phosphate buffer, supplemented with 50 mM NaCl, 10 mM EDTA, pH 6.0), a stock solution of 10 mM TCEP was added until a final TCEP concentration of 1 mM in the case of antibodies and 0.5 mM in the case of antibody fragments. Incubation was carried out at room temperature for 60 min with gentle stirring. The reducing agent was removed by centrifugation through Amicon Ultra-4 filters (maximum throughput 10 kDa), or using Zeba Spin Desalting Column, 7K MWCO (Thermo Fisher Scientific, USA). Then to the solution of antibodies or antibody fragments were added the corresponding solutions of mc-vc-PAB-MMAE (or mc-vc-PAB-MMAF) in various molar ratios. Two variants of buffers for site-directed reactions were used: phosphate buffer - 20 mM phosphate buffer, supplemented with 50 mM NaCl, 10 mM EDTA, pH 6.0 or borate buffer, 1 mM EDTA, 250 mM NaCl, pH 8.0. The incubation lasted 2 h at room temperature with gentle stirring on a shaker. To purify ADCs from the unreacted low molecular weight component (mc-vc-PAB-MMAE or mc-vc-PAB-MMAF), we used Zeba Spin Desalting Column, 7K MWCO, then the conjugates were sterilized by filtration through a 0.22 μm membrane. Protein concentration was determined spectrophotometrically at a wavelength of 280 nm using a BioDrop μLITE instrument.

Preparation of conjugates of antibodies / antibody fragments with cytotoxic drug DM1.

Another format for ADCs was the conjugate of the cytotoxic preparation of maytansinoid DM1 and an antibody (antibody fragment) linked by a non-cleavable linker succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC). This bifunctional linker connects to the cysteine DM1 due to the maleimide group, and to the amino groups of the lysine of the antibody fragment due to the thioether group. Upon entering the lysosomes, DM1 is released from ADCs, which is an effective antimitotic agent that causes cell death even at very low concentrations.

Conjugates of antibodies / antibody fragments with SMCC-DM1 were prepared in two ways.

1) In the first case, GD2-specific antibodies / antibody fragments were transferred into a buffer containing 50 mM potassium phosphate and 2 mM EDTA, pH 7.0. SMCC (MedChemExpres, USA) was dissolved in DMSO and added to the protein solution to a final SMCC / Ab molar ratio of 10: 1. The reaction was carried out for 3 hours at room temperature with stirring. The MCC-modified antibody (antibody fragment) was then purified on a Zeba Spin Desalting Column, 7K MWCO, equilibrated in 35 mM sodium citrate with 150 mM NaCl and 2 mM EDTA, pH 6.0. DM1 dissolved in DMSO was added to the MCC-antibody compound (antibody fragment), in a molar ratio of DM1 to protein of 10: 1. The reaction was carried out for 4-20 hours at room temperature with stirring.

2)In the second case, GD2-specific antibodies / antibody fragments were transferred into a buffer containing 35 mM sodium citrate with 150 mM NaCl and 2 mM EDTA, pH 6.0. Conjugate SMCC-DM1 (MedChemExpres, USA) dissolved in DMSO was added to the antibodies / antibody fragments, in a molar ratio of DM1 to protein of 10: 1. The reaction was carried out for 4-20 hours at room temperature with stirring.

In both cases, to purify ADCs from the unreacted low-molecular-weight component, we used Zeba Spin Desalting Column, 7K MWCO, then the conjugates were sterilized by filtration through a 0.22 μm membrane. Protein concentration was determined spectrophotometrically at a wavelength of 280 nm using a BioDrop μLITE instrument.

Evaluation of the binding specificity of the obtained ADCs with ganglioside GD2 in ELISA.

Ganglioside GD2 was dispensed into the wells of a 96-well Greiner ELISA-plate microlon HB in 50 μl ethanol. The final ganglioside concentration was 0.1 μg / well. The sorption of ganglioside was carried out for 18 h at a temperature of 4 ° C. Plugging of nonspecific binding sites was carried out in 2% BSA solution in PBS for 1 hour at 37 ° C. The wells were washed in PBS-T (PBS + Tween 0.05%) 3 times on an Autowash II automatic washer (Labsystems, Spain). Various dilutions of ADCs based on GD2-specific antibodies or their fragments in PBS-T were added to the wells. Incubation with antibodies / fragments lasted for at least 2 hours at 37 ° C. After 3 washes in PBS-T, anti-species HRP-labeled antibodies or anti-FLAG HRP-labeled antibodies (titer 1: 6000) were dropped into the wells. One hour after incubation at 37 ° C and three washes in PBS-T, 100 μl of developer (substrate-chromogenic mixture TMB, Immunotech or 1-Step Ultra TMB-ELISA Solution) was added to the wells. After the development of color, the reaction was stopped by adding 50 μl of 10% H ₂ SO ₄ to the wells. The optical density was measured at 450 nm on a Multiscan FC plate spectrophotometer (Thermo scientific, USA).

Staining of cells of GD2-positive and GD2-negative lines with GD2-specific antibodies for flow cytometry.

Cells of GD2-positive and GD2-negative lines were washed 2 times in PBA (PBS supplemented with 1% FBS, 0.02% NaN ₃ ). Staining with AlexaFluor488-labeled anti-GD2 antibodies or their fragments was performed in 100 μl for 1 hour at 4 ° C. After two washes in PBS, the sample volume was brought to 0.5 ml and transferred to cytometric tubes for subsequent measurements on an EPICS ELITE flow cytometer (Coulter, USA). In each sample, at least 5 × 10 ³ cells were recorded. The results were processed using the WinMDI program.

Study of the cytotoxic effects of GD2-specific antibodies and their fragments.

The MTT test was performed using a standard colorimetric procedure [Denizot F. J. ImMunol. Methods. 1986.]. The cells were incubated with ADCs for 72 h, then centrifuged (8 min, 300 g), the pellet was suspended, and 30 μl / well of MTT solution (5 mg / ml in PBS) was added. After the crystals of formazan had precipitated (2-4 h), it was dissolved by the addition of 100 μl of DMSO. Optical absorption was measured on a Multiscan FC spectrophotometer (ThermoScientific, USA) at a wavelength of 540 nm. The plotting was performed using the SigmaPlot, MS Excel software. The results presented are either the average of at least three independent experiments.

Evaluation of the antitumor effects of ADCs in a syngeneic GD2-positive cancer model.

EL-4 cells (1 * 10 ⁷ ) were injected subcutaneously into C57BL / 6 mice (females aged 12 to 14 weeks). When the tumors reached the size of about 200 mm ³ , the mice were randomly divided into 3 groups (in each group, 5 mice). In the first experimental group, mice received intravenous injections of chimeric GD2-specific antibodies 14.18 (100 μg / mouse or 4 μg / kg), in the second experimental group, mice received intravenous injections of ADCs ch14.18-MMAF (100 μg / mouse or 4 μg / kg ). In the control group, mice received intravenous injections of saline. There were 4 injections with an interval of 4 days.

Example 2. Purification of chimeric GD2-specific antibodies and scFv antibody fragments.

Analysis of the expression and purification of chimeric GD2-specific antibodies 14.18, as well as their scFv-fragments was performed by protein electrophoresis in 12% polyacrylamide gel in the presence of SDS under reducing conditions (Fig. 1 A, B). The scFv fragments were further analyzed by immunoblotting using antibodies specific for the FLAG-tag (Fig. 1B). On electrophoresis of antibodies, 2 protein bands are visible corresponding to the light and heavy chains of immunoglobulins (Fig. 1 A), and for antibody fragments - one band of about 27 kDa, which corresponds to the calculated mass of the scFv fragment. This protein also effectively binds anti-FLAG antibodies (Fig. 1B), which confirms the presence of the FLAG-tag included in the construct. After purification, the secreted protein was presented mainly in monomeric form, despite the fact that the terminal cysteine was included in the structure of the molecule. At the same time, in order to prevent possible dimerization of fragments with “unclosed” terminal cysteines, exogenous closing of the terminal cysteine was performed by adding oxidized glutathione. The production and purity of the isolated chimeric GD2-specific antibodies and scFv fragments were at a fairly high level.

Example 3. Creation of conjugates of chimeric GD2-specific antibodies and scFv fragments with cytotoxic drugs MMAE and MMAF.

To obtain ADCs, a maleimide-activated linker carrying MMAE or MMAF was conjugated with thiol groups of cysteine bridges in the case of chimeric antibodies or with cysteine-terminal scFv fragments. The general structure of an antibody-MMAE conjugate is shown in FIG. 2.

The molar ratios of antibodies / antibody fragments and constructs carrying MMAE (or MMAF) were selected, as well as the optimal buffer for site-directed reaction. As a result of the selection of conditions, there were, on average, 4 molecules of cytotoxic substance per antibody molecule (n = 4), and only one molecule of the drug (n = 1) bound to the scFv fragment molecule, which was determined by the design of the fragment construction, which included one terminal cysteine ... FIG. 3A shows the results of electrophoretic separation of chimeric antibodies 14.18 (Ch14.18), scFv fragments of 14.18, and antibody-drug conjugates. The conjugation of the linker carrying the cytotoxic compound MMAE with antibodies and antibody fragments did not lead to a significant change in molecular weight, which is expected due to the low molecular weight of the construct (1330 Da) and the attachment of 1 to 4 such molecules per 1 antibody molecule or antibody fragment. Conjugation did not lead to the formation of aggregates or, on the contrary, to protein degradation. However, the linkage of such conjugates could affect the antigen-binding characteristics of full-length GD2-specific antibodies and scFv fragments. Therefore, the binding of the resulting ADCs to the GD2 ganglioside was evaluated in comparison with the parent GD2 binding molecules. The direct ELISA results are shown in FIG. 3B-D.

As seen in FIG. 3, site-directed conjugation of MMAE and MMAF preparations did not significantly affect the ability of GD2-binding molecules to interact with ganglioside GD2, and it can be concluded that this approach for obtaining ADCs is justified and promising, since antibodies 14.18, as well as their scFv fragments, retain antigen-binding ability after carrying out modifications on the thiol groups of the protein. It was also shown that for all variants of the obtained ADCs the specificity did not change and there was no cross-reactivity with other gangliosides (GD3, GM2, and GD1b) even at high concentrations of conjugates.

Example 4. Creation of conjugates of chimeric GD2-specific antibodies and scFv fragments with the cytotoxic drug DM1.

The general structure of an antibody-DM1 conjugate is shown in FIG. 4A. In this case, DM1 is attached randomly at the amino groups of the lysines that make up the antibody or antibody fragment. In the structure of antibodies 14.18, lysines are present both in constant parts and in variable domains. Cross-linking of drug molecules in the antigen-binding region of an antibody can reduce or completely prevent interaction with the antigen. Therefore, the selection of the optimal molar ratios of the DM1 drug to the protein was an important task, especially in the case of scFv antibody fragments, since they only include antibody variable domains. As a result of the selection of the conditions, there were on average 4-5 molecules of cytotoxic substance per antibody molecule, and a maximum of 1-2 DM1 molecules bound to the scFv fragment without a significant decrease in the interaction with the antigen. For multimeric fragments of antibodies, as a result of the selection of conditions, it was possible to increase the number n to 4-5. Evaluation of the binding of the resulting ADCs to the GD2 ganglioside compared to the parent GD2 binding molecules is shown in FIG. 4B, C and D.

Example 5. Cytotoxic effects of ADCs in in vitro experiments .

At the first stage, several GD2-positive and -negative cell lines were selected. FIG. 5 shows the cytometric staining of human neuroblastoma IMR-32, murine EL-4 lymphoma and human neuroblastoma NGP-127 cells with GD2-specific antibodies, which shows that the NGP-127 line is GD2-negative, and the IMR-32 and EL-4 lines - GD2-positive, with GD2 expression on EL-4 cells higher than on IMR-32 cells.

From the presented data, it can be seen that ADCs containing drugs MMAE or MMAF induce a significant level of cell death only in cells of lines carrying GD2, and do not act on GD2-negative cells in the selected concentration range (Fig. 5A, B). The concentration inhibiting cell survival by 50% (IC50%) for the EL-4 line was 65 pM in the case of incubation with ch14.18-MMAF (including full-length chimeric antibodies) and 310 pM in the case of incubation with ch14.18-MMAE (Fig. AT). These very low IC50% values are likely a synergistic effect of the cytotoxic chemotherapy and GD2-specific antibodies, which have their own direct cytotoxic activity. For EL-4 cells, the conjugate with the MMAF preparation was found to be more effective than the ADC with MMAE. This pattern was typical for both GD2-specific antibodies and scFv fragments 14.18. In general, the cytotoxic activity of scFv fragments was significantly lower than full-length antibodies, which is probably due to the lower affinity of the fragments for GD2 and the fact that only one molecule of the cytotoxic drug is included in the scFv-based conjugate, while the ADC of the full-length antibody contains on average 4 drug molecules. At the same time, the production of ADCs based on antibody fragments has significant potential and prospects, since it is this antibody format that can improve the therapeutic index of antibody-drug conjugates by improving pharmacokinetic characteristics and reducing side effects. We have obtained multimeric pegylated antibody fragments that combine the advantages of full-length antibodies and monovalent antibody fragments. Their circulation time in the blood and their affinity are comparable or even superior to full-length antibodies, they are free of Fc-mediated side effects and are characterized by better accumulation in the tumor due to their smaller size. In further work, the pegylated fragments will be used by us to create ADCs, along with the optimization of ADCs based on full-length antibodies.

ADCs containing the drug DM1 also induce a significant level of cell death only in cell lines carrying GD2 with little effect on GD2 negative cells in the selected concentration range (Fig. 7). ADCs based on antibodies with conjugated DM1 induce cell death with a slightly lower efficiency compared to ADCs carrying MMAE and MMAF. The cytotoxic activity of ADCs containing monomeric scFv fragments was found to be similar for both drug options.

Multimerization of antibody fragments led to an increase in the antigen-binding ability of antibody fragments and, potentially, to an increase in the cytotoxic properties of molecules. At the same time, size and weight are important parameters for the manifestation of the antitumor effects of therapeutic molecules. For example, molecules with a mass of more than 150 kDa have limited potential in the clinic due to their inability to pass through the walls of blood vessels. In this regard, multimers of antibody fragments with a molecular weight of more than 150 kDa were not used in further work. They did not fall under this limitation and were analyzed in subsequent cytotoxic tests of mono-, di- and tetra-scFv-PEG, as well as mono- and di-Fab-PEG.

It was shown in the work that ADCs with multimeric fragments of antibodies induce cell death of tumor cells significantly more strongly than ADCs based on monomeric fragments (Fig. 7).

Example 6. Antitumor effects of ch14.18-MMAF in a syngeneic GD2-positive cancer model.

Since the EL-4 cell line was obtained from C57BL / 6 mice and is characterized by a high level of expression of the GD2 ganglioside, these cells were used to create a syngeneic cancer model to study the antitumor effects of the created ADCs. Tumor cells were injected subcutaneously into mice. For the experiment, we chose the conjugate of the full-length antibody ch14.18 with the cytotoxic drug MMAF, since it showed the maximum activity in in vitro experiments . As a control, 2 groups of mice were created, which, after the development of the tumor, were intravenously injected with the initial “naked” antibodies or saline as a negative control (Fig. 8).

The growth of tumors in mice was extremely aggressive and characterized by exponential growth. This is probably due to the high proliferation of EL-4 cells, as in culture the doubling of these cells occurs in 8 hours, although for most cell lines the cell cycle is about 18-20 hours. In this experiment, the antitumor effect of the initial antibodies was observed, but it was insignificant and, in general, did not affect the development of the tumor process. In contrast to the original antibodies, the antibody-drug conjugate significantly slowed down tumor growth in this group of mice, and complete elimination of the tumor was recorded in one mouse of this group. In addition, it should be noted that no obvious toxic effects were observed in healthy mice on the administration of ADCs at a concentration of 10 μg / kg.

Thus, in the present invention, GD2-specific antibodies and scFv fragments of GD2-specific antibodies were developed and purified. Techniques were refined and antibody / antibody-drug conjugates (ADCs) were prepared. The cytotoxic effects of the obtained ADCs were assessed, which showed their high selectivity and cytotoxic effectiveness. Experiments carried out on a syngeneic cancer model showed unexpected and significant antitumor effects of the obtained ADCs immunoconjugates, which opens up prospects for the use of these immunoconjugates for the therapy of GD2-positive oncological diseases.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are provided for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

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LIST OF SEQUENCES

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Claims (13)

1. Immunoconjugate for the treatment of GD2-positive oncological diseases, having the general formula A- (L-C) n, where (a) A is an antibody or antigen-binding antibody fragment that specifically binds ganglioside GD2, or a multimer of antibody fragments that includes one to four antigen-binding antibody fragments, each of which specifically binds ganglioside GD2, having a heavy chain containing H-CDR1 with SEQ ID NO: 1, H-CDR2 with SEQ ID NO: 2 and HCDR3 with SEQ ID NO: 3, and a light chain containing L-CDR1 with SEQ ID NO: 4, L-CDR2 with SEQ ID NO : 5 and L-CDR3 with SEQ ID NO: 6; (b) L is a linker; (c) C is a cytotoxic or cytostatic agent; and (d) n ranges from 1 to 8. 2. Immunoconjugate according to claim 1, characterized in that the heavy chain contains a variable domain with a sequence homologous to at least 95% of the sequence of SEQ ID NO: 7. 3. Immunoconjugate according to claim 1, characterized in that the light chain contains a variable domain with a sequence homologous to at least 95% of the sequence of SEQ ID NO: 8. 4. Immunoconjugate according to claim 1, characterized in that the cytotoxic or cytostatic agent is selected from the group of dolastin derivatives or maytansinoids. 5. Immunoconjugate according to claim 4, characterized in that the cytotoxic or cytostatic agent is selected from the group: monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, maytansinoid DM4 or maytansinoid DM1. 6. Immunoconjugate according to claim 1, characterized in that A is a multivalent construct containing two, three or four antigen-binding antibody fragments, each of which specifically binds ganglioside GD2, linked together by covalent conjugation with a polyethylene glycol molecule. 7. Composition for the treatment of a GD2-specific tumor disease, containing at least an immunoconjugate according to any one of claims. 1-6 in a therapeutically effective amount, as well as a pharmaceutically acceptable carrier, diluent and / or excipient. 8. A composition according to claim 7, characterized in that the GD2-specific tumor disease is neuroblastoma, glioma, small cell lung cancer, breast cancer or sarcoma. 9. A method of treating a GD2-specific tumor disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to claim 7.

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