Anti-GD2 Antibodies, Immunoconjugates, and Therapeutic Uses Thereof

Abstract

The present invention provides an antibody that binds to human GD2 protein, as well as an isolated nucleic acid and a host cell comprising a sequence encoding the antibody.The present invention also provides an immunoconjugate comprising the antibody linked to a growth inhibitory agent, and a pharmaceutical composition comprising the antibody or immunoconjugate of the present invention.The present invention also provides the use of the antibody, immunoconjugate, and pharmaceutical composition of the present invention for the purpose of treating or diagnosing cancer.

Description

[Technical field]

[0001] The present invention relates to an antibody that binds to human GD2 (disialoganglioside GD2), as well as isolated nucleic acids and host cells comprising sequences encoding said antibody. The present invention also relates to an immunoconjugate comprising said antibody linked to a growth inhibitory agent, and a pharmaceutical composition comprising the immunoconjugate of the invention. The present invention also relates to the use of the antibody, immunoconjugate, and pharmaceutical composition of the invention for therapeutic and / or diagnostic purposes of cancer. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are a class of therapeutics that combine the specificity of monoclonal antibodies (mAbs) with the potency of cytotoxic molecules. The use of ADCs enhances the cancer-killing activity of antibodies with conjugated cytotoxic agents while reducing systemic toxicity caused by exposure to free toxic agents through target-specific delivery. Currently, a total of 12 ADCs have been approved by the FDA to treat human cancers, most of which were approved in the last 2-3 years. ADCETRIS® (brentuximab vedotin or SGN-35), an anti-CD30 antibody conjugated with the cytotoxic agent MMAE, designed to treat CD30-positive recurrent lymphoma, and KADCYLA® (T-DM1), an anti-HER2 antibody conjugated with the cytotoxic agent DM1, designed to treat HER2-positive metastatic breast cancer, were the first two approved ADCs. These initial approvals have been followed by at least eight more approvals.

[0003] Linker technology has a great impact on the efficacy, specificity, and safety of ADCs. Enzyme-cleavable linkers exploit the difference in intracellular and extracellular protease activity to achieve controlled drug release. Drugs can be conjugated to antibodies by many different linkers and can be specifically cleaved only by the action of lysosomal proteases that are present intracellularly and at high levels in certain tumor types (Koblinsk et al, 2000). This ensures the stability of the linker in the bloodstream and limits damage to healthy tissues. However, the associated increased hydrophobicity of some enzyme-labile linkers can cause ADC aggregation, especially for highly hydrophobic drugs. Thus, there is a need for linkers that can provide increased serum stability and solubility, allowing efficient conjugation and intracellular delivery of hydrophobic drugs.

[0004] This document provides examples of anti-GD2 monoclonal antibodies in the treatment of neuroblastoma and other oncology indications. Currently, three anti-GD2 naked antibody drugs are approved for human use: naxitamab (Danyelza®) and dinutuximab (Unituxin®) in the United States, and dinutuximab beta (Qarziba®) in Europe. Dinutuximab and dinutuximab beta are mouse-human chimeric antibodies (ch14.18) produced in mouse myeloma cell lines called SP2 / 0 cells and CHO (Chinese Hamster Ovary) cells, respectively, while naxitamab is a humanized antibody (hu3F8) produced by CHO cells.

[0005] Approximately 12% of all pediatric cancer patients die from neuroblastoma, the most common extracranial solid tumor in childhood. The majority of patients are diagnosed with high-risk neuroblastoma, which has a mortality rate of 50%. Treatment of high-risk neuroblastoma consists of multimodality therapy with significant short-term and long-term toxicities. Recurrence rates are high and further treatment options are limited. Intensification of conventional therapy has not improved prognosis and is instead associated with increased toxicity.

[0006] As mentioned above, dinutuximab, an antibody against ganglioside GD2, a glycan-containing sphingolipid antigen that is uniformly expressed in neuroblastoma, cancers of neuroectodermal origin, and neural tissue, has been approved by the FDA for the treatment of neuroblastoma. Application of this antibody in combination with cytokines and differentiation factors has improved patient prognosis and demonstrated that neuroblastoma is sensitive to immunotherapy (Yu et al., 2010; New Engl. J. Med, Vol 363: pp 1324-34; and Suzuki and Cheung., 2015; Expert Opin Ther Targets Vol 19: p. 349-62). Dinutuximab improved event-free survival compared with previous standard chemotherapy treatments. Nevertheless, the favorable clinical outcomes observed with administration of anti-GD2 antibody alone can sometimes be accompanied by severe toxicity. Although numerous toxicities have been documented (e.g., tachycardia, hypertension, hypotension, fever, and urticaria), by far the most debilitating toxicity is neuropathic pain. In fact, despite the concomitant use of powerful analgesics (including opioids), this neuropathic pain often limits the dose of anti-GD2 antibodies, thereby limiting their effectiveness. This neuropathic pain is believed to be mediated by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] Furthermore, progress has been made with the introduction of disialoganglioside (GD2) immunotherapy in patients with high-risk (>12 months) or refractory / recurrent neuroblastoma (Ahmed et al., 2014, Nazha et al., 2020). However, neuropathic pain is a common and significant dose-limiting adverse event that prevents patients from being fully treatable with currently marketed GD2 antibodies (dinutuximab, naxitamab). Binding of anti-GD2 antibodies to GD2 expressed on peripheral somatosensory and visceral nerves mediates severe acute pain and / or allodynia at various body sites in patients (Yuki et al., 1997) and monkeys (EMA / 263814 / 2017, 2017) and appears to be associated with peripheral nerve injury. Despite the relatively low expression of GD2 on peripheral nerves (Slart et al., 1997; Lammie et al., 1993; Yuki et al., 1997; Vriesendorp et al., 1997), effector functions of the Fc portion of antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which should also kill tumor cells, are thought to induce immunological attack on peripheral nerves by humoral and cellular mediators, manifesting as neuropathy and acute neuropathic pain (Sorkin 2002; Dobrenkov and Cheung 2014; Mastrangelo et al., 2020; Nazha et al., 2020).

[0008] Another example of an ADC that induces peripheral neuropathy is monomethyl auristatin E (MMAE), a potent anticancer microtubule targeting agent (MTA), which is used as the payload of three approved MMAE-containing ADCs (and several ADCs currently in clinical development) for treating various types of cancer. MMAE-ADCs often induce peripheral neuropathy, a frequent adverse event that leads to the reduction or interruption of the therapeutic dose of many MMAE-ADCs and their subsequent clinical discontinuation. MMAE-ADC-induced peripheral neuropathy is due to nonspecific uptake of ADCs and release of MMAE in peripheral nerves, which disrupts microtubules (MTs) and causes neurodegeneration.

[0009] Thus, what is needed is an anti-GD2 ADC, the antibody of which preferably has reduced CDC and ADCC, is conjugated to a non-neurotoxic cytotoxic payload (due in part to its chemical makeup and formulation) and can more selectively deliver its cytotoxic payload to malignant cells without causing toxicity, particularly peripheral neuropathy, that would otherwise limit dosing and therapeutic efficacy. Summary of the Invention

[0010] The humanized anti-GD2 antibody hu14.18 binds to disialoganglioside GD2, which is found on the cell surface of tumors, including neuroblastoma, melanoma, sarcoma, etc., and on normal tissues that are primarily restricted to the central and peripheral nervous systems in humans. Expression of disialoganglioside GD2 in normal tissues is essentially restricted to the central nervous system, peripheral sensory nerve fibers, skin melanocytes, lymphocytes, and mesenchymal stem cells. Treatment with anti-GD2 antibodies can induce peripheral neuropathy and pain, which is thought to be related to antibody effector functions such as complement fixation and complement-dependent cytotoxicity (CDC) that affect peripheral nerves. Previously, a hu14.18 antibody was developed with a point mutation K322A (Kabat EU index numbering) in the immunoglobulin IgG1 constant domain, designated "hu14.18-IgG1(K322A)" (described in U.S. Patent Application Publication No. 8,835,606(B2) and incorporated herein by reference). This K322A point mutation in the hu14.18 antibody was designed to prevent activation of the complement cascade and CDC effector functions while still maintaining the potential for antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. In addition, hu14.18-IgG1(K322A) was produced with hypofucosylation to enhance ADCC activity (described in U.S. Patent Application Publication No. 8,835,606(B2) and all amino acid and nucleic acid sequences are incorporated herein by reference). ADCC-enhanced hu14.18-IgG1(K322A) may induce less complement fixation / CDC-mediated pain while killing tumor cells via the ADCC mechanism of action. ADCC-enhanced hu14.18-IgG1(K322A) is in clinical development (phase 2) but is not approved. A chimeric anti-GD2 antibody with a wild-type IgG1 isotype (dinutuximab) has been approved for a defined set of pediatric neuroblastoma patients, and this treatment is known to cause neuropathic pain and may require coadministration with pain-relieving medications.

[0011] In some embodiments, the present invention describes anti-GD2 hu14.18 antibodies as ADCs with reduced or absent antibody effector functions of both ADCC and CDC, thereby providing the advantage of a larger therapeutic window for treating unmet medical needs. Designing hu14.18 antibodies without significant ADCC or CDC effector functions may reduce the risk of neuropathic pain induced by administration of anti-GD2 hu14.18 antibodies, while conjugation of drugs to the antibodies provides a different mechanism of action of such anti-GD2-ADCs to eliminate tumor cells. The combination of anti-GD2 hu14.18 lacking significant ADCC and CDC effector functions and delivering a potent conjugated drug as an ADC is likely to improve existing treatments and meet the (currently) unmet medical needs of patients. In selected embodiments, the selected cytotoxic drug must be one that does not have neurotoxic properties.

[0012] Binding of an antibody to a specific antigen target is a property of the antigen-binding fragment (Fab) domain, which contains specific light and heavy chain variable region complementarity determining region (CDR) amino acid sequences. Antibody effector functions, as well as binding to Fc gamma receptors (FcγR) and neonatal Fc receptors (FcRn), are properties of immunoglobulin (Ig) constant domains. Different immunoglobulin constant domain genes exist, and these constant domain genes fused together with the Fab domain sequence determine the antibody isotype (IgM, IgG1, IgG2, etc.). In naturally occurring or recombinant antibodies, the same antigen-binding domain amino acid sequence can be fused and expressed to different constant domain isotypes to produce antibodies with the same antigen target binding specificity, but with different effector function properties. The ability of an antibody to mediate the activation of components of the immune system is referred to as the "effector function" of the antibody, and important effector functions are mediated through binding to FcγR on immune cells and complement proteins such as complement component 1q (C1q) in blood and extracellular fluids. Depending on the isotype, antibodies can link specific antigen targets on cells to the immune system and induce directed attacks on target cells by immune functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Binding of Ig constant domains to FcRn plays an important role in the in vivo half-life of antibodies through increased recycling back to the circulation after the antibody is internalized in cells. Numerous studies have been conducted to test the role of specific amino acid residues in antibody constant domains for antibody effector functions and antibody binding to FcγR and complement proteins. Using some changes in the amino acid sequence of human Ig constant domains, recombinant antibodies with altered properties for antibody effector functions and antibody binding to FcγR and complement proteins can be produced. In selected embodiments, the invention provides ADC proteins that bind to target GD2 and deliver ADC molecules to cells without significant antibody effector functions.

[0013] In some embodiments of the present invention, the production and use of anti-GD2 hu14.18-IgG1.4(K322A)-delK antibodies conjugated to small molecule toxic payloads from the class of exatecant topoisomerase I inhibitors for the treatment of different oncology indications, such as sarcoma, neuroblastoma and SCLC, is described. This novel ADC offers a favorable therapeutic window compared to other GD2 antibody therapies already in clinical use.

[0014] In a preferred embodiment of the invention, the Fc moiety effector function was engineered to be removed from the ADC and instead, a non-neurotoxic payload, exatecan, was used to kill tumor cells, resulting in the absence of PNS injury and obvious clinical pain signals in rats and monkeys after repeated injections of molecule 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition "GD2" is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastomas and melanomas, with expression in normal tissues highly restricted to the central and peripheral nervous systems in humans. GD2 is defined by the following chemical structure: [ka] This corresponds to the IUPAC name: (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-hydro oxy-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.

[0016] As used herein, the name "M4344" refers to an adenosine triphosphate (ATP) competitive inhibitor having the following chemical structure: [ka]

[0017] A "domain" or "region" can be any region of a protein that is generally defined based on sequence homology and often associated with a particular structural or functional entity. GD2 family members are known to be composed of Ig-like domains. The term domain is used herein to indicate either an individual Ig-like domain, such as the "N-domain," or a group of consecutive domains, such as the "A2-B2 domain."

[0018] A "coding sequence" or a sequence that "encodes" an expression product such as a polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0019] As used herein, reference to a particular protein (e.g., an antibody) can include a polypeptide having a native amino acid sequence, as well as variants and modified forms, regardless of their origin or mode of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as that obtained in nature. Such native sequence proteins may be isolated from nature or may be prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternatively spliced ​​forms), naturally occurring allelic variants, and forms that include post-translational modifications. Native sequence proteins include proteins that have post-translational modifications, such as glycosylation, or phosphorylation, or other modifications of some amino acid residues.

[0020] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. Some genes that are not structural genes may be transcribed from DNA into RNA, but may not be translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may contemplate a genomic sequence that codes for a protein, i.e., a sequence that includes regulatory elements, promoter, intron and exon sequences.

[0021] As used herein, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the reference sequence. Thus, the percentage of "sequence identity" can be determined by comparing two such sequences over their entire length by global pairwise alignment using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), for example, using the Needle program (EMBOSS) with the BLOSUM62 matrix and the following parameters: gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10, end gap extend=0.5 (these are standard settings).

[0022] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups can also be defined based on amino acid size.

[0023] An "antibody" (also called an "immunoglobulin") may be, for example, a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) that determine aspects of the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains distinct sequence domains (or regions). The light chain of a typical IgG antibody contains two regions, a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody contains four regions, a variable region (VH) and a constant region (CH), the latter being composed of three constant domains (CH1, CH2, and CH3). The variable regions of both the light and heavy chains determine the binding and specificity to the antigen. The constant regions of the light and heavy chains can confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an antibody and consists of the variable portions of one light chain and one heavy chain.

[0024] The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is mainly composed of residues from the so-called hypervariable regions or complementarity determining regions (CDRs). Complementarity determining regions (CDRs) therefore refer to amino acid sequences that together define the binding affinity and specificity of the Fv region of an antibody. The light (L) and heavy (H) chains of an antibody each have three CDRs, which are called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen binding site of a conventional antibody contains six CDRs, including a set of CDRs derived from each of the heavy and light chain variable regions.

[0025] "Framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., the portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the framework regions of naturally occurring human antibodies.

[0026] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody molecule of a single amino acid sequence directed against a particular antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody can be produced, for example, by a single clone of a B cell or hybridoma, but can also be produced recombinantly, including by methods such as genetic engineering or protein engineering.

[0027] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises the VH and VL of an antibody from a non-human animal combined with the CH and CL of another antibody, which in some embodiments is a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also refer to multispecific antibodies that have specificity for at least two different antigens.

[0028] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and that has been modified, for example by replacing certain amino acids in the VH and VL framework regions, to avoid or minimize an immune response in humans. The constant regions of a humanized antibody are typically human CH and CL regions.

[0029] A "fragment" of an antibody (e.g., a conventional antibody) comprises a portion of an intact antibody, such as IgG, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a heavy chain antibody or a single domain antibody, such as a VHH.

[0030] The term "Fab" refers to an antibody fragment in which about half of the N-terminal side of the heavy chain and the entire light chain are bound to each other via disulfide bonds, and which has a molecular weight of about 50,000 Da and an antigen-binding activity. Fab is usually obtained from fragments of IgG by treating it with the protease papain.

[0031] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen-binding activity, which is slightly larger than two identical Fab fragments linked by disulfide bonds in the hinge region. F(ab')2 is usually obtained from fragments of IgG by treating it with the protease pepsin.

[0032] The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0033] Single chain Fvs ("scFvs") are covalently linked VH:VL heterodimers, usually expressed from gene fusions containing genes encoding VH and VL linked via a peptide-encoded linker. Human scFv fragments of the invention contain the CDRs held in the proper conformation, for example by using recombinant gene techniques. Bivalent and multivalent antibody fragments may form spontaneously by association of monovalent scFvs, or may be generated by coupling monovalent scFvs via a peptide linker, such as bivalent sc(Fv)2. A "dsFv" is a VH:VL heterodimer stabilized by a disulfide bond. "(dsFv)2" means two dsFvs coupled via a peptide linker.

[0034] An "antibody drug conjugate" or "ADC" is an antibody conjugated to one or more cytotoxins, each via a linker. The antibody is typically a monoclonal antibody specific for an antigen. In certain preferred embodiments of the present invention, ADCs are designed as targeted therapy for treating cancer. Unlike chemotherapy alone, these preferred embodiments combine the targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, which can distinguish between healthy and malignant tissues.

[0035] As used herein, "molecule 1" (also referred to herein as "M3554") refers to the following ADC: [ka] wherein the antibody binds to GD2 and comprises the amino acid sequence of SEQ ID NO:4 and the amino acid sequence of SEQ ID NO:1, the linker is a β-glucuronide, and the proliferation inhibitor is exatecan.

[0036] The term "hybridoma" refers to a cell that produces a desired monoclonal antibody having antigen specificity, obtained by cell fusion between a B cell prepared by immunizing a non-human mammal with an antigen and a myeloma cell derived from a mouse or the like. In some instances, the name "M3554" is synonymous with molecule 1.

[0037] "Purified" or "isolated," when referring to a polypeptide (e.g., an antibody) or nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified," as used herein, means that at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of the same type of biological macromolecules are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide, although the molecule may contain certain additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0038] As used herein, the term "subject" means a mammal, such as a rodent, cat, dog, primate, or human. In embodiments of the invention, the subject (or patient) is a human.

[0039] As used herein, the term "peripheral neuropathy" means damage to the nerves located outside the brain and spinal cord (peripheral nerves), manifested clinically as weakness, numbness, and / or pain.

[0040] As used herein, "neuropathic pain" refers to pain caused by damage or injury to the nerves that carry information from the skin, muscles, and other parts of the body between the brain and spinal cord, and is manifested clinically as a burning sensation and often hypersensitivity to touch in the affected area.

[0041] Methods for Producing Antibodies of the Invention The antibodies of the present invention can be produced by any of the techniques known in the art, including but not limited to any chemical, biological, genetic or enzymatic techniques, or a combination thereof.

[0042] By knowing the amino acid sequence of the desired antibody, one of skill in the art can readily produce said antibody or immunoglobulin chains using standard techniques for the production of polypeptides. For example, they can be synthesized using the well-known solid-phase method using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems, Foster City, California) following the manufacturer's instructions. Alternatively, the antibodies and immunoglobulin chains of the present invention can be produced by recombinant DNA technology as is well known in the art. For example, these polypeptides (e.g., antibodies) can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired polypeptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, which can then be isolated using well-known techniques.

[0043] The amino acid sequences of the antibody chains are used to generate protein precursors of anti-GD2-ADC molecules. The mature anti-GD2 hu14.18 light and heavy chain amino acid sequences are shown. The C-terminus of the hu14.18 heavy chain can end at either G446 (Kabat EU index numbering) or K447 (Kabat EU index numbering) as shown. Changes in the constant domains from the wild-type human IgG1 sequence are underlined. hu14.18 mature light chain amino acid sequence: [Table 1] hu14.18-IgG1-delK mature heavy chain amino acid sequence: [Table 2]

[0044] hu14.18-IgG1.4-delK mature heavy chain amino acid sequence: [Table 3] hu14.18-IgG1.4(K322A)-delK heavy chain amino acid sequence [Table 4]

[0045] Flowchart providing the candidate characterization cascade used to test anti-GD2 antibody protein precursor proteins: [ka]

[0046] The present invention further relates to a method for producing an antibody of the present invention, comprising the steps of: (i) culturing a transformed host cell according to the present invention; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0047] Antibodies of the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0048] In some embodiments, the humanized chimeric antibody of the present invention can be produced by obtaining nucleic acid sequences encoding the humanized VL and VH regions as described above, inserting them into an expression vector for animal cells carrying genes encoding a human antibody CH and a human antibody CL to construct a human chimeric antibody expression vector, and expressing the coding sequences by introducing the expression vector into an animal cell.

[0049] As the CH domain of a human chimeric antibody, any region belonging to a human immunoglobulin heavy chain can be used, for example, one of the IgG class is suitable, and any one of the subclasses belonging to the IgG class, for example, IgG1, IgG2, IgG3, and IgG4, can be used. Furthermore, as the CL of a human chimeric antibody, any region belonging to a human immunoglobulin light chain can be used, and one of the kappa class or lambda class can be used.

[0050] Methods for producing humanized or chimeric antibodies can involve conventional recombinant DNA and gene transfection techniques, which are well known in the art (see, for example, Morrison SL. et al. (1984) and patent documents U.S. Pat. Nos. 5,202,238 and 5,204,244).

[0051] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art including, for example, the techniques disclosed in WO 2009 / 032661, CDR-grafting (EP 239,400; PCT WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA.et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application No. 125023 and WO 96 / 02576).

[0052] The Fab of the present invention can be obtained by treating an antibody (e.g., IgG) of the present invention with a protease, such as papain, or can be produced by inserting DNA sequences encoding both chains of the Fab of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic cell (whichever is appropriate) to express the Fab.

[0053] The F(ab')2 of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with pepsin, which is a protease. In addition, F(ab')2 can also be produced by linking Fab', which will be described later, via a thioether bond or disulfide bond.

[0054] The Fab' of the present invention can be obtained by treating the F(ab')2 of the present invention with a reducing agent, such as dithiothreitol. Alternatively, the Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic cell (whichever is appropriate) to effect expression.

[0055] The scFv of the present invention can be produced by obtaining the sequences of the CDRs or VH and VL domains as previously described for the antibodies of the present invention, then constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic cell (whichever is appropriate) to express the scFv. A well-known technique called CDR grafting may be used to generate humanized scFv fragments, which involves selecting complementarity determining regions (CDRs) according to the present invention and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, for example, WO 98 / 45322; WO 87 / 02671; U.S. Pat. Nos. 5,859,205; 5,585,089; 4,816,567; EP 0173494).

[0056] Modified anti-GD2 antibody Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0057] Modifications and variations can be made to the structure of the antibodies of the invention and the DNA sequences encoding them and still result in a functional antibody or polypeptide possessing desirable characteristics.

[0058] When making changes to the amino acid sequence of a polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index to the interactive biological function of a protein is generally understood in the art. It is accepted that the relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics, and these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0059] Further aspects of the present invention also include function-conservative variants of the polypeptides of the invention.

[0060] For example, certain amino acids in a protein structure can be substituted with other amino acids without significant loss of activity. Because the interaction ability and properties of a protein define its biological functional activity, certain amino acid substitutions can be made in a protein sequence and, of course, in its coding DNA sequence, and still obtain a protein with similar properties. Therefore, it is contemplated that various changes can be made in the antibody sequence of the present invention, or the corresponding DNA sequence encoding the above polypeptide, without significant loss of their biological activity.

[0061] It is known in the art that certain amino acids can be substituted by other amino acids with similar hydropathic indexes or scores and still result in proteins with similar biological activity, i.e., biologically functionally equivalent proteins. Using well-established techniques such as alanine scanning techniques, it is also possible to identify all amino acids that can be substituted in the antibodies or polypeptides of the present invention without significant loss of antigen binding. Such residues can be identified as neutral because they are not involved in antigen binding or maintaining antibody structure. One or more of these neutral positions can be substituted by alanine or another amino acid without changing the main characteristics of the antibodies or polypeptides of the present invention.

[0062] Neutral positions can be considered as positions that can incorporate any amino acid substitution. In fact, in the principle of alanine scanning, alanine is selected because this residue does not have any particular structural or chemical characteristics. It is generally accepted that if a certain amino acid can be substituted with alanine without changing the properties of a protein, then it is likely that many, if not all, other amino acid substitutions are also neutral. In the reverse case, where alanine is the wild-type amino acid, if a certain substitution is neutral, it is likely that other substitutions are also neutral.

[0063] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account any of the above characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0064] It may be desirable to modify the antibodies of the present invention with respect to effector function, for example to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or to alter, for example, binding to Fc receptors. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, a cysteine ​​residue(s) can be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. Homodimeric antibodies thus generated may exhibit improved internalization capability and / or increased complement-mediated cell killing and / or increased antibody-dependent cellular cytotoxicity (ADCC) (Caron PC.et al.1992; and Shopes B.1992). In some embodiments, the antibodies of the present invention may be antibodies with modified amino acid sequences that result in reduced or eliminated binding to most Fcγ receptors, which may reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages, liver sinusoidal cells, etc.

[0065] Another type of amino acid modification of the antibodies of the invention, by deleting one or more carbohydrate moieties found in the antibody and / or by adding one or more glycosylation sites that are not present in the antibody, may be useful to alter the original glycosylation pattern of the antibody. The presence of either of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, creates a potential site for glycosylation. Addition or deletion of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites).

[0066] Another type of modification involves the removal of sequences identified, either in silico or experimentally, that may result in degradation products or heterogeneity of antibody formulations. As an example, deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites, particularly Asn-Gly, are present in an antibody or polypeptide, removal of the site may be considered, typically by a conservative substitution to remove one of the involved residues. Such substitutions in the sequence to remove one or more of the involved residues are also intended to be encompassed by the present invention.

[0067] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N-linked or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO 87 / 05330.

[0068] Removal of carbohydrate moieties present on an antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment cleaves most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and Edge, A. S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved using a variety of endo- and exoglycosidases as described in Thotakura, N. R. et al. (1987).

[0069] Another type of covalent modification of an antibody involves linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods described, for example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, and 4,179,337.

[0070] Other amino acid sequence modifications known in the art can also be applied to the antibodies of the invention.

[0071] Anti-GD2 ADC The present invention provides immunoconjugates, also referred to herein as ADCs or more simply as conjugates.As used herein, these terms all have the same meaning and are interchangeable.The immunoconjugates of the present invention can be prepared according to the in vitro methods described herein.

[0072] The invention provides ADCs comprising an antibody of the invention (such as mAb1, or an antibody having similar CDRs to mAb1) covalently linked via a linker to at least one growth inhibitory agent.

[0073] The term "growth inhibitory agent" (also called "anti-proliferative agent") refers to a molecule or compound or composition that inhibits the growth of cells, such as tumor cells, in vitro and / or in vivo.

[0074] In some embodiments, the growth inhibitory agent is a cytotoxic drug (also called a cytotoxic agent).The present invention also contemplates radioactive moieties for use as cytotoxic agents.

[0075] The term "cytotoxic agent," as used herein, refers to a substance that directly or indirectly inhibits or prevents the function of a cell and / or causes the destruction of a cell. The term "cytotoxic agent" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomaymycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatins or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 and CC-1065 analogs.

[0076] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in changing the topology of DNA by catalyzing the transient breakage and rejoining of single strands of DNA.Topoisomerase I inhibitors are highly toxic, for example, to dividing mammalian cells.Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, and rubitecan.

[0077] In some embodiments, the immunoconjugates of the invention comprise the cytotoxic drug exatecan as a growth inhibitory agent. Exatecan has the following IUPAC chemical name:

[0078] (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione. Exatecan has the following structure: [ka] It is expressed by:

[0079] Exatecan is a modified derivative of camptothecin that has an additional alicyclic ring fused to ring A and ring B that contains a solubilizing primary amine (corresponding to the 7-CH2NH2 substituent on camptothecin). There are also lipophilic substituents at positions 10 and 11 on ring A that enhance membrane permeability.

[0080] In further embodiments of the invention, other CPT analogs and other cytotoxic drugs may be used, such as those listed above. Examples of some cytotoxic drugs and conjugation methods are further provided in WO 2008 / 010101, which is incorporated by reference.

[0081] The term "radioactive moiety" means At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89 Radioisotopes generally primarily emit beta radiation. In some embodiments, the radioisotope is an alpha-emitting isotope, such as thorium-227, which emits alpha radiation. Immunoconjugates can be prepared, for example, as described in WO 2004 / 091668.

[0082] In the immunoconjugate of the present invention, the antibody of the present invention is covalently linked to at least one growth inhibitory agent via a linker. "Linker" as used herein means a chemical moiety that includes a covalent bond and / or any chain of atoms that covalently attaches the growth inhibitory agent to the antibody. Linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Conjugation of the antibodies of the invention with cytotoxic or other growth inhibitory agents can be accomplished using, for example, bifunctional derivatives of the following: N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio)propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imidoesters, This can be done using a variety of bifunctional protein coupling agents, including but not limited to conductors (e.g., dimethyl adipomidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0083] In embodiments of the present invention, the linker may be a "cleavable linker," which may facilitate the release of a cytotoxic drug or other growth inhibitory agent in or near a cell, such as a tumor cell. In some embodiments, the linker is a linker that is cleavable in the endosome of a mammalian cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) may be used.

[0084] The following nomenclature is also used herein when referring to structural formulas representing immunoconjugates: the growth inhibitory agent and the linker are collectively also referred to as the [(linker)-(growth inhibitory agent)] moiety, e.g., the exatecan molecule and the linker are collectively also referred to as the [(linker)-(exatecan)] moiety.

[0085] In some particular embodiments of the invention, the linker is a linker cleavable by the human enzyme glucuronidase. Thus, for example, an immunoconjugate of the invention may have the following formula (II) which includes a linker cleavable by glucuronidase: [ka] It may have

[0086] where the antibody is an antibody of the invention, S is a sulfur atom of the antibody, and n is the number of [(linker)-(growth inhibitor)] moieties covalently linked to the antibody. The number n can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​of the antibody. In some embodiments, the antibody is mAb1.

[0087] The number n is also referred to as the "drug-to-antibody ratio" (or "DAR"). This number n should always be understood to be an average number for any given (preparation of) immunoconjugate.

[0088] In other specific embodiments of the invention, the linker is a linker cleavable by the human enzyme legumain. Thus, for example, an immunoconjugate of the invention may have the following formula (III) which includes a linker cleavable by legumain: [ka] It may have

[0089] where the antibody is an antibody of the invention, S is a sulfur atom of the antibody, and n is the number of [(linker)-(growth inhibitor)] moieties covalently linked to the antibody. The number n (also called DAR) can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​of the antibody. In some embodiments, the antibody is mAb1.

[0090] In each of the above formulas (II) and (III), the chemical structure between the sulfur atom of the antibody and the growth inhibitory agent is a linker, one of which is also included in each of the formulas (IV)-(IX), which are further illustrated below.

[0091] In any one of the embodiments described above having a glucuronidase or legumain cleavable linker, the growth inhibitory agent can be, for example, exatecan.

[0092] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, the immunoconjugate having the following formula (IV): [ka] having

[0093] wherein S is a sulfur atom of an antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0094] In another embodiment, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, the immunoconjugate having the following formula (V): [ka] having

[0095] wherein S is a sulfur atom of an antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0096] In some embodiments, in the immunoconjugates of the invention, such as the exatecan conjugates having glucuronidase-cleavable or legumain-cleavable linkers described above, the linker is covalently attached to the antibody at the sulfur atom of a cysteine ​​residue of the antibody. For example, this cysteine ​​residue of the antibody may be one of the cysteine ​​residues capable of forming an interchain disulfide bond (also referred to herein as an interchain disulfide bridge). Since an IgG1 antibody has four interchain disulfide bonds with a total of eight cysteine ​​residues, attaching a linker to the antibody at the sulfur atom of such a cysteine ​​residue results in a DAR that may be up to 8, in such cases the DAR is typically 7-8, such as 7.5-8.0 (i.e., about 8), provided that the antibody is an IgG1 or has the same number of interchain disulfide bonds as an IgG1.

[0097] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, the immunoconjugate having the following formula (VI): [ka] having

[0098] wherein S is a sulfur atom of a cysteine ​​of the antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10, in more specific embodiments, n is 7 to 8, and in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8).

[0099] In another embodiment, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, the immunoconjugate having the following formula (VII): [ka] having

[0100] wherein S is a sulfur atom of a cysteine ​​of the antibody and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10, in more specific embodiments, n is 7 to 8, and in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8).

[0101] Any of the antibodies of the invention (described hereinabove and below) can be used in any of the above immunoconjugates. In some embodiments, the immunoconjugates of the invention comprise mAb1 as the antibody.

[0102] Thus, in some embodiments, the present invention provides an immunoconjugate comprising mAb1 covalently linked to exatecan via a linker, the immunoconjugate having the following formula (VIII): [ka] having The immunoconjugate is provided in which S is a sulfur atom of a cysteine ​​of the antibody mAb1, and n is the number of [(linker)-(exatecan)] moieties covalently linked to mAb1. The number n (also referred to as DAR) can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​of mAb1 capable of forming an interchain disulfide bridge, and the DAR is about 8. Examples of such immunoconjugates (i.e., "ADC1") are further described in the Examples. In another embodiment, the present invention provides an immunoconjugate comprising mAb1 covalently linked to exatecan via a linker, the immunoconjugate having the following formula (IX): [ka] having

[0103] The immunoconjugate is provided in which S is a sulfur atom of a cysteine ​​of antibody mAb1, and n is the number of [(linker)-(exatecan)] moieties covalently linked to mAb1. The number n (also referred to as DAR) can be, for example, 1-10, in more specific embodiments, n is 7-8, and in even more specific embodiments, n is 7.5-8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​of mAb1 that can form an interchain disulfide bridge, and the DAR is about 8. The number of cytotoxic and / or cytostatic agents linked to the antigen-binding portion of the anti-huLRRC15 ADC can vary (referred to as the "drug-antibody ratio" or "DAR"), limited only by the number of available attachment sites on the antigen-binding portion and the number of agents linked to a single linker. Anti-GD2 ADCs with a DAR of 20 or more are contemplated, so long as the anti-GD2 ADCs do not exhibit unacceptable levels of aggregation under the conditions of use and / or storage.

[0104] In other embodiments of the invention, the linker may be a "non-cleavable linker" (e.g., an SMCC linker). Release of the growth inhibitory agent from the antibody may occur upon lysosomal degradation of the antibody.

[0105] In other embodiments of the invention, the immunoconjugate may be a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide (as a growth inhibitory agent); such fusion proteins may be produced by recombinant techniques or by peptide synthesis, i.e., by methods well known in the art. The molecule of coding DNA may contain respective regions encoding the two parts of the conjugate (the antibody and the cytotoxic or growth inhibitory polypeptide, respectively), either adjacent to each other or separated by a region encoding a linker peptide.

[0106] In some embodiments of the invention, pain was reduced by avoiding CDC through an Fc point mutation (K322A) in the hu14.18 antibody while maintaining sufficient ADCC activity to attack tumor cells, as supported by experimental data from a rat allodynia model showing partial alleviation of mechanical allodynia (Slart et al., 1997; Sorkin et al., 2010).

[0107] To this end, an ADC (molecule 1) was designed to induce tumor cell apoptosis after ADC internalization and intracellular enzymatic release of the payload in lysosomes by conjugating a humanized ch14.18-derived antibody with exatecan, a potent DNA topoisomerase I inhibitor. In addition, both ADCC and CDC activities were engineered to be removed from the Fc portion of the antibody to attenuate neuronal damage by these mechanisms. Because topoisomerase I inhibitors, in contrast to microtubule inhibitors (Stagg et al., 2016), do not induce peripheral neuropathy by themselves (Verschraegen et al., 2000, Rowinsky 2005) or as part of an ADC (Ogitani et al., 2016), cell killing of relatively high GD2-expressing tumors by the payload was achieved while sparing the low GD2-expressing peripheral nervous system (PNS), thereby reducing the pain effect.

[0108] The antibodies of the invention can also be used in directed enzyme prodrug therapy, such as antibody-directed enzyme prodrug therapy, by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active cytotoxic drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278). The enzyme component of an immunoconjugate useful for ADEPT can include any enzyme capable of acting on a prodrug to convert it into its more active, cytotoxic form. Enzymes useful in this context include, but are not limited to, alkaline phosphatases, useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, useful for converting the non-toxic fluorocytosine to the anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (e.g., cathepsins), useful for converting peptide-containing prodrugs to free drugs. tyrosinase B and L); D-alanyl carboxypeptidases, useful for converting prodrugs containing D-amino acid substituents; glycosylation enzymes, such as O-galactosidase and neuraminidase, useful for converting glycosylated prodrugs to the free drug; P-lactamases, useful for converting P-lactam derivatized drugs to the free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at the amine nitrogen with a phenoxyacetyl or phenylacetyl group, respectively, to the free drug. Enzymes can be covalently attached to the antibodies of the invention by techniques well known in the art, such as the use of linkers as described above.

[0109] Suitable methods for preparing the immunoconjugates of the present invention are well known in the art (see, for example, Hermanson GT, Bioconjugate Techniques, Third Edition, 2013, Academic Press). For example, methods are well known for conjugating cytotoxic drugs to antibodies via linkers that are covalently attached to cysteine ​​residues of the antibody interchain disulfide bridges.

[0110] Generally, the immunoconjugates of the invention comprise, for example: (i) preparing a compound comprising a linker and a growth inhibitory agent (e.g., a cytotoxic drug), also referred to herein as a "drug-linker compound"; (ii) contacting an optionally buffered aqueous solution of an antibody according to the invention with a solution of a drug-linker compound; (iii) then optionally separating the conjugate formed in (ii) from unreacted antibody and / or drug-linker compound. The method can be obtained by a method comprising the steps of:

[0111] The aqueous solution of the antibody may be buffered with a buffer such as, for example, histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer may be selected depending on the nature of the antibody. The drug-linker compound may be dissolved in an organic polar solvent such as, for example, dimethylsulfoxide (DMSO) or dimethylacetamide (DMA).

[0112] For conjugation with cysteine ​​residues of the antibody, the antibody is subjected to reduction (e.g., using TCEP) prior to step (ii). Suitable reducing conditions for reducing only interchain disulfide bonds are known in the art.

[0113] The reaction temperature of the conjugation is usually 20-40°C. The reaction time can vary, typically 1-24 hours. The reaction between the antibody and the drug-linker compound can be monitored by size exclusion chromatography (SEC) using a refractometer and / or a UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0114] Those skilled in the art can use many different chromatographic methods to carry out the separation of step (iii).The conjugate can be purified, for example, by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography, such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC), such as reversed-phase HPLC.Purification by dialysis or filtration or diafiltration can also be used.

[0115] After steps (ii) and / or (iii), the conjugate-containing solution may be subjected to an additional purification step (iv), for example by chromatography, ultrafiltration, and / or diafiltration. Such additional purification steps, for example by chromatography, ultrafiltration, and / or diafiltration, can also be performed on the antibody-containing solution after the reduction reaction, if reduction is performed before conjugation.

[0116] The conjugate is recovered in aqueous solution at the end of such process. Drug-to-antibody ratio (DAR) is a value that can vary depending on the nature of the antibody and drug-linker compound used as well as the experimental conditions used for conjugation ((drug-linker compound) / (antibody) ratio, reaction time, nature of the solvent and co-solvent if present, etc.). Thus, contacting the antibody with the drug-linker compound can result in a mixture containing several conjugates that differ from each other by different drug-to-antibody ratios. The determined DAR is therefore an average value.

[0117] Using an antibody with four interchain disulfide bridges (e.g., mAb1 or any IgG1 antibody) and performing conjugation at the cysteine ​​residues of the interchain disulfide bridges is a method well known in the art and offers the advantage that by choosing reaction conditions that allow the conjugation to proceed to completion (or at least approach completion), a relatively uniform DAR of approximately 8 can be achieved.

[0118] An exemplary method that can be used to determine the DAR is to measure the λ D and 280 nm, a wavelength commonly used to measure protein concentrations, such as antibody concentrations. D is selected to allow for discrimination between the drug and the antibody, i.e., λ D is the wavelength at which the drug has high absorbance, and λ D is far enough away from 280 nm to avoid substantial overlap of the absorbance peaks of the drug and antibody. D can be selected to be 370 nm for exatecan (or camptothecin or other camptothecin analogues) or 252 nm for maytansinoid molecules.

[0119] The method of DAR calculation can be derived, for example, from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science: D (A λD ) and 280 nm (A 280 The absorbance of the conjugate at 300 nm is measured either by size exclusion chromatography (SEC) analysis of the monomer peak (allowing for the calculation of the "DAR(SEC)" parameter) or by using classical spectrophotometer equipment (allowing for the calculation of the "DAR(UV)" parameter). The absorbance can be expressed as: A λD =(C D ×ε DλD )+(C A ×ε AλD ) A 280 =(C D ×ε D280 )+(C A ×ε A280 ) In the formula, C D and C A are the concentrations of drug and antibody in solution, respectively. ε DλD and ε D280 are λ D and the molar extinction coefficient of the drug at 280 nm. ε AλD and ε A280 are λ D and the molar extinction coefficient of the antibody at 280 nm.

[0120] Solving these two equations in two unknowns gives us the following: C D =[(ε A280 ×A λD )-(ε AλD ×A 280 )] / [(ε DλD ×ε A280 )-(ε AλD ×ε D280 )] C A =[A 280 -(C D ×ε D280 )] / ε A280 The average DAR is then calculated from the ratio of drug concentration to antibody concentration: DAR=C D / C A . Exemplary methods for preparing the immunoconjugates of the invention are described in the Examples.

[0121] Drug-Linker Compounds The present invention also provides compounds comprising a linker and a growth inhibitory agent (e.g., a cytotoxic drug), also referred to herein as "drug-linker compounds." For example, the present invention provides compounds of formula (X): [ka] or a pharma- ceutically acceptable salt thereof, which compound is also referred to herein as "Drug-Linker Compound 1," "Compound DL1," or "DL1." The present invention also relates to a compound of formula (XI): [ka] or a pharma- ceutically acceptable salt thereof, which compound is also referred to herein as "Drug-Linker Compound 2," "Compound DL2," or "DL2."

[0122] These drug-linker compounds can be used to prepare the immunoconjugates of the invention described herein above and below.

[0123] Drug-Linker Compounds of the invention (eg, those of formula (X) or (XI) depicted above) can be prepared by chemical synthesis, for example, as further described in the Examples below.

[0124] Pharmaceutical Compositions The antibodies or immunoconjugates of the invention can be combined with pharma- ceutically acceptable carriers, diluents, and / or excipients, and optionally sustained release matrices, including but not limited to types of biodegradable polymers, non-biodegradable polymers, lipids, or sugars, to form pharmaceutical compositions.

[0125] Accordingly, another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the invention and a pharma- ceutically acceptable carrier, diluent, and / or excipient.

[0126] "Pharmaceutical" or "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to a mammal, optionally, particularly a human. A pharmaceutically acceptable carrier, diluent, or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0127] As used herein, "pharmaceutical acceptable carriers" include any solvents, dispersion media, coatings, antibacterial agents, and antifungal agents that are physiologically compatible. Examples of suitable carriers, diluents, and / or excipients include, but are not limited to, one or more of water, amino acids, saline, phosphate buffered saline, phosphate buffer, acetate buffer, citrate buffer, succinate buffer; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts such as NaCl or calcium chloride; sugars or polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, and the like; and combinations thereof. In many cases, it is useful to include an isotonic agent such as a sugar, polyhydric alcohol, or sodium chloride in the pharmaceutical composition, and the formulation may also include an antioxidant such as tryptamine and / or a stabilizer such as Tween 20.

[0128] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will necessarily depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0129] The pharmaceutical compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0130] In one embodiment, the pharmaceutical composition comprises a pharma- ceutically acceptable vehicle for injection preparations.The pharma-ceutically acceptable vehicle may be an isotonic sterile saline solution (such as monosodium phosphate or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), or a dried, particularly lyophilized, composition that allows the constitution of an injection solution upon addition of sterile water or saline, as the case may be.

[0131] The pharmaceutical composition can be administered via a drug combination device.

[0132] The dose used for administration can be adjusted as a function of various parameters, for example, depending on the mode of administration used, the pathology involved, or the desired duration of treatment.

[0133] To prepare pharmaceutical compositions, an effective amount of the antibody or immunoconjugate of the invention may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0134] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions; in all such cases the form must be sterile, must be injectable using an appropriate device or system for delivery without degradation, and must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0135] The solution of the active compound as free base or pharmacologically acceptable salt can be prepared in water, preferably mixed with surfactant.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil.Under normal conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0136] The antibody or immunoconjugate of the present invention can be formulated into a pharmaceutical composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, and the like.

[0137] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it may be desirable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent, optionally with any of the other ingredients listed above, followed by filtration sterilization.Generally, dispersion can be prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients required from those listed above.In the case of sterile powder for preparing sterile injectable solution, preparation method includes vacuum drying and freeze-drying technology, which can obtain powder of active ingredient and any additional desired ingredient from its solution that has been previously sterilized and filtered.

[0139] The preparation of more or more highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as a solvent is envisioned to result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0140] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0141] For example, for parenteral administration in an aqueous solution, the solution may be suitably buffered if necessary, and the liquid diluent may first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that may be used will be known to those skilled in the art in light of this disclosure. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the intended injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject.

[0142] The antibodies or immunoconjugates of the invention can be formulated into therapeutic mixtures containing, for example, about 0.01 to 100 milligrams per dose.

[0143] In addition to antibodies or immunoconjugates formulated for parenteral administration, such as intravenous or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solids for oral administration, sustained release capsules, and any other forms currently in use.

[0144] In some embodiments, the use of liposomes and / or nanoparticles is contemplated for introducing the polypeptide into the host cell. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0145] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (size of about 0.1 μm) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles or biodegradable polylactide or polylactide-co-glycolide nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily made by those skilled in the art.

[0146] Liposomes are formed from phospholipids dispersed in an aqueous medium and can spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). The diameter of MLVs is generally between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 A, containing aqueous solution in their core. The physical properties of liposomes vary depending on pH, ionic strength, and the presence of divalent cations.

[0147] In addition to the examples mentioned above, further pharmaceutical forms such as nanoparticles, microparticles and microcapsules, implants (eg, lipid implants), or self-solidifying or self-emulsifying systems are also contemplated.

[0148] Treatment Methods and Uses The inventors have found that the antibodies of the invention (e.g., mAb1) can be internalized as part of a GD2-antibody complex after binding. Furthermore, target-mediated uptake of such antibodies conjugated to a cytotoxic drug (in a preferred embodiment, exatecan) has been shown to lead to increased in vitro cytotoxicity potency in GD2-expressing tumor cells compared to the respective control ADC or target-negative cells. The inventors have also shown that these immunoconjugates of the invention elicit significant antitumor activity in vivo when used at doses ranging from 0.25 mg / kg to 10 mg / kg, in single or multiple injections. Indeed, the immunoconjugates of the invention show broad activity in a large set of in vitro and in vivo models derived from different tumor types. Furthermore, the immunoconjugates of the invention were well tolerated in dose-ranging studies in non-human primates. These preclinical data indicate a good therapeutic window for subsequent clinical trials. Thus, the antibodies, immunoconjugates, and pharmaceutical compositions of the invention may be useful for treating cancers expressing GD2.

[0149] Thus, the present invention provides an antibody, immunoconjugate, or pharmaceutical composition of the present invention for use as a medicament. For example, the present invention provides an antibody, immunoconjugate, or pharmaceutical composition of the present invention for use in treating cancer. The present invention further provides a method for treating cancer comprising administering to a subject in need thereof an antibody, immunoconjugate, or pharmaceutical composition of the present invention.

[0150] The cancer to be treated with the antibody, immunoconjugate, or pharmaceutical composition of the invention is preferably a cancer that expresses GD2, and more preferably a cancer that overexpresses GD2 compared to normal (i.e., non-tumor) cells of the same tissue origin. Expression of GD2 by cells can be readily assayed, for example, by using an antibody according to the invention (or a commercially available anti-GD2 antibody), for example as described in the "Diagnostic Uses" section below, and for example by immunohistochemistry.

[0151] GD2 is involved in tumor development and malignant phenotype through enhanced cell proliferation, motility, migration, adhesion, and invasion, depending on the tumor type. This provides a rationale for targeting disialoganglioside GD2 in cancer therapy for any tumor type that overexpresses GD2. Although the present invention is not limited to a particular mechanism of action, known anti-GD2 monoclonal antibodies target GD2-expressing tumor cells, leading to phagocytosis and destruction by antibody-dependent cell-mediated cytotoxicity, lysis by complement-dependent cytotoxicity, and apoptosis and necrosis by direct induction of cell death. In addition, anti-GD2 monoclonal antibodies can also prevent homing and adhesion of circulating malignant cells to the extracellular matrix.

[0152] In some embodiments, the cancer treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the invention is neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, and soft tissue sarcoma. In selected embodiments, the cancer treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the invention is neuroblastoma and osteosarcoma.

[0153] The antibodies or immunoconjugates of the invention may be used alone or in combination with any suitable growth inhibitory agent in the treatment of cancer.

[0154] The antibody of the present invention may be conjugated (linked) to a growth inhibitory agent, as described above. Thus, the antibody of the present invention may be useful for targeting the growth inhibitory agent to cancerous cells that express or overexpress GD2 on their surface.

[0155] It is also well known that therapeutic monoclonal antibodies can result in the depletion of cells bearing the antigen specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent cytolysis, and direct inhibition of tumor growth by signals mediated by the antigen targeted by the antibody.

[0156] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, can be performed.

[0157] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies bound to their cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al. (Journal of Immunological Methods. 1997 Mar;202(2):163-171) can be performed.

[0158] In some embodiments, the antibodies of the invention may be antibodies with altered amino acid sequences that result in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues that express such receptors, e.g., macrophages, liver sinusoidal cells, etc.

[0159] Aspects of the invention relate to a method for treating cancer, comprising administering a therapeutically effective amount of an antibody, immunoconjugate, or pharmaceutical composition of the invention to a subject in need thereof.

[0160] In the present invention, the term "treat" or "treatment", as used herein, means to improve, alleviate, inhibit the progression of, or prevent the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treat cancer", as used herein, means to inhibit the proliferation of malignant cells of a tumor and / or the progression of metastases from said tumor. Such treatment may also result in the regression of tumor growth, i.e., a measurable reduction in the size of the tumor. For example, such treatment may result in the complete regression of tumors or metastases.

[0161] In the context of therapeutic applications of the present invention, the term "subject" or "patient" or "subject in need thereof" or "patient in need thereof" refers to a subject (e.g., a human or non-human mammal) that is affected or likely to be affected by a tumor. For example, the patient may be a patient who has been determined to be susceptible to a therapeutic agent targeting GD2, in particular an antibody or immunoconjugate according to the present invention, e.g., by the methods described herein below.

[0162] By "therapeutically effective amount" is meant an amount sufficient to treat the above-mentioned cancer disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the antibodies, immunoconjugates, and pharmaceutical compositions of the present invention (collectively referred to as "therapeutic agents") will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific therapeutic agent used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific therapeutic agent used; the duration of treatment; drugs used in combination or simultaneously with the specific therapeutic agent used; and similar factors well known in the medical art. For example, it is well known to those skilled in the art to start with a dose of the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0163] The antibodies, immunoconjugates, or pharmaceutical compositions of the invention may also be used to inhibit the progression of cancer metastasis.

[0164] The antibodies, immunoconjugates, or pharmaceutical compositions of the invention can also be used in combination with any other therapeutic intervention (e.g., adjuvant therapy) for treating cancer and / or reducing the growth of metastatic cancer. For example, the other therapeutic intervention for such combination can be a standard of care (SOC) therapeutic agent for the cancer to be treated.

[0165] The efficacy of treatment with an antibody or immunoconjugate or pharmaceutical composition according to the invention can easily be assayed in vivo, for example in a mouse model of cancer, by measuring, for example, the change in tumor volume, the percentage of tumor regression, the percentage of partial regression, or the percentage of complete regression between treatment and control groups.

[0166] Diagnostic Use GD2 has been reported to be highly expressed on the surface of cancer cells, such as neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, soft tissue sarcoma, or other solid tumors that express GD2.

[0167] Therefore, GD2 can be considered a cancer marker and has the potential to be used to indicate the effectiveness of anti-cancer treatment or to detect disease recurrence.

[0168] In one embodiment, the antibodies of the invention can be used as a component of an assay to determine a patient's sensitivity to a therapeutic agent in a treatment targeting GD2-expressing tumors, to monitor the effectiveness of an anti-cancer treatment, or to detect disease recurrence after treatment. In some embodiments, the same antibody of the invention can be used as both a component of a therapeutic agent and a component of a diagnostic assay.

[0169] Therefore, a further aspect of the present invention relates to the use of the antibody according to the present invention for detecting GD2 expression in a biological sample from a subject ex vivo. Another aspect of the present invention relates to the use of the antibody according to the present invention for detecting GD2 expression in a subject in vivo. When used to detect GD2, the antibody can be labeled with a detectable molecule, such as a fluorophore or an enzyme.

[0170] Detection of GD2 By detecting the expression of surface GD2 on tumor cells, a) diagnosing the presence of cancer in a subject; or b) determining the sensitivity of a patient with cancer to a therapeutic agent targeting GD2, in particular an antibody or immunoconjugate according to the invention; or c) monitoring the efficacy of an anti-GD2 cancer treatment or detecting cancer recurrence after an anti-GD2 cancer treatment, in particular said treatment being a treatment with an antibody or immunoconjugate according to the invention;

[0171] may be intended.

[0172] In some embodiments, the antibody of the present invention is intended for diagnostic use in vitro or ex vivo.For example, GD2 can be detected in a biological sample obtained from a subject using the antibody of the present invention in vitro or ex vivo.The use according to the present invention can also be an in vivo use.For example, the antibody of the present invention can be administered to a subject, and antibody-cell complexes can be detected and / or quantified, where detection of said complexes indicates cancer.

[0173] The present invention provides an in vitro or ex vivo method for detecting the presence of cancer in a subject, comprising: (a) contacting a biological sample derived from a subject with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibody bound to the biological sample; (c) detecting the presence of cancer by comparing the measured level of bound antibody to a control, where an increased level of bound antibody compared to the control is indicative of cancer. The present invention further relates to a method comprising the steps of:

[0174] The present invention also relates to an in vitro or ex vivo method for determining the sensitivity of a patient suffering from cancer to a therapeutic agent targeting GD2, in particular an antibody or immunoconjugate according to the present invention, comprising: (a) contacting a biological sample derived from a patient with cancer with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibody bound to the biological sample; (c) comparing the measured level of bound antibody to the biological sample with the level of bound antibody in a control;

[0175] An increase in the level of bound antibody to said biological sample compared to a control indicates that the patient is susceptible to a therapeutic agent that targets GD2.

[0176] In the above methods, the control may be a normal, non-cancerous biological sample of the same type, or may be a reference value determined to be indicative of the level of antibody binding in a normal biological sample of the same type.

[0177] In one embodiment, the antibodies of the invention are useful for diagnosing GD2-expressing cancers, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express GD2.

[0178] The present invention provides an in vitro or ex vivo method for monitoring the efficacy of an anti-GD2 cancer treatment, comprising: (a) contacting a biological sample from a subject undergoing anti-GD2 cancer treatment with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibody bound to the biological sample; (c) comparing the measured level of bound antibody to the level of bound antibody in a control;

[0179] The present invention further relates to a method, wherein a decrease in the level of binding antibody to the biological sample compared to the control indicates that the anti-GD2 cancer treatment is effective. In the method, an increase in the level of binding antibody to the biological sample compared to the control indicates that the anti-GD2 cancer treatment is not effective. In one embodiment of this method for monitoring efficacy, the control is the same type of biological sample as the biological sample subjected to analysis, but is obtained from the subject at an earlier time point during the course of anti-GD2 cancer treatment.

[0180] The present invention provides an in vitro or ex vivo method for detecting cancer recurrence following anti-GD2 cancer treatment, comprising: (a) contacting a biological sample from a subject who has completed an anti-GD2 cancer treatment with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibody bound to the biological sample; (c) comparing the measured level of bound antibody to the level of bound antibody in a control;

[0181] The method further relates to a method in which an increase in the level of binding antibodies to said biological sample compared to a control indicates cancer recurrence after said anti-GD2 cancer treatment. Said control may in particular be a biological sample of the same type as the biological sample subjected to analysis, but obtained from the subject before, i.e. at or after completion of anti-GD2 cancer treatment.

[0182] The anti-GD2 cancer treatment is, for example, a treatment using an antibody or immunoconjugate according to the invention, which targets GD2-expressing cancers such as neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, and soft tissue sarcoma, or other solid tumors expressing GD2.

[0183] In some embodiments, the antibodies of the present invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule or fluorophore, a radioactive molecule, an enzyme, or any other label known in the art that provides (directly or indirectly) a signal.

[0184] As used herein, the term "labeled" with respect to an antibody according to the invention is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the polypeptide, and indirect labeling of the polypeptide by reactivity with a detectable substance.

[0185] The antibodies of the present invention can be labeled with a radioactive molecule by any method known in the art. For example, the radioactive molecule can be I 123 , I 124 , In 111 , Re 186 , Re 188 , Tc 99 Examples of radioactive atoms include, but are not limited to, radioactive atoms for scintigraphic studies, such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0186] "Biological sample" encompasses various sample types obtained from a subject that can be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. Thus, biological samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples such as tumor samples.

[0187] In some embodiments, the biological sample may be a formalin-fixed and paraffin-embedded (FFPE) or frozen tissue sample.

[0188] The present invention also provides an in vivo method for detecting the presence of cancer in a subject, comprising: a) administering to a patient an antibody according to the invention labeled with a detectable molecule; b) detecting localization of said antibody in the patient by imaging, e.g., by detecting a detectable molecule. The present invention relates to a method comprising the steps of:

[0189] In the above methods, the cancer can be a GD2-expressing cancer such as neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, and soft tissue sarcoma, or other solid tumors that express GD2.

[0190] The antibodies of the invention may also be useful in staging cancer (e.g., in radioimaging).The antibodies of the invention may be used alone or in combination with other cancer markers.

[0191] The terms "detection" or "detected" as used herein include qualitative and / or quantitative detection (ie, measuring the levels) with or without reference to a control.

[0192] In the present invention, the term "diagnose" as used herein means determining the nature of a medical condition based on some collected data, with the intention of identifying the pathology affecting a subject.

[0193] kit Finally, the present invention also provides a kit comprising at least one antibody or immunoconjugate of the present invention. The kit comprising the antibody of the present invention can be used for detecting surface protein GD2 or for therapeutic or diagnostic assays. The kit of the present invention can comprise an antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., sepharose beads). A kit can be provided that comprises an antibody for detecting and quantifying surface protein GD2 in vitro, for example, by ELISA or Western blot. Such antibodies useful for detection can be provided with a label, such as a fluorescent label or a radioactive label. A brief description of the sequence Amino acid sequence: Primary structure of the hu14.18-IgG1.4(K322A)-delK antibody: Heavy chain sequence (SEQ ID NO:4): [Table 5] Light chain sequence (SEQ ID NO:1): [Table 6]

[0194] Preferred embodiments of anti-GD2 ADCs In a preferred embodiment, the present invention describes new ADC drugs that are fully optimized according to the following outline. Selection of antibodies with appropriate affinity, good cell binding ability, and efficient internalization to ensure sufficient payload kinetics after antibody binding to cancer cells. The selected antibody, hu14.18-IgG1.4(K322A)-delK, is based on the known CDR sequence of dinutuximab (ch14.18), but is further optimized by humanization to reduce the immunogenicity risk in patients.

[0195] The antibody portion contains the HC K322A mutation that eliminates complement activation, which reduces pain side effects, as demonstrated by a pilot study of a humanized anti-GD2 monoclonal antibody (hu14.18K322A) in combination with other drugs, including chemotherapy.

[0196] The antibody portion is sequence modified to nearly eliminate binding to most Fcγ receptors (IgG1.4 format). Preclinical tolerability studies in non-human primates (detailed below) demonstrated that this modification, together with the K322A mutation, is primarily responsible for the nearly complete reduction in pain effects and nerve tissue damage. Thus, as a potential therapeutic agent, such IgG1.4 modifications in ADCs may reduce uptake and associated toxicity in other normal cells and tissues expressing Fcγ receptors (e.g., macrophages and liver sinusoidal cells).

[0197] In a preferred embodiment, the antibody moiety is conjugated to the highly potent topoisomerase-I inhibitor exatecan to improve antitumor activity compared to naked anti-GD2 antibody treatment. In contrast to other ADC payloads such as tubulin toxins (e.g., auristatins and maytansines), this preferred ADC has a lower risk of associated neurotoxicity. This is particularly advantageous when the ADC target is presented on the surface of peripheral nerves. (5) The linker connecting the drug and the antibody is designed to maximize systemic stability after parenteral application. Furthermore, the payload-linker moiety based on a glucuronide linkage system ensures good conjugation, favorable PK properties, and reduced nonspecific uptake in target-negative cells and tissues. (6) The exatecan payload of the ADC used in the present invention has demonstrated efficacy in combination with DNA damage response inhibitors, such as ATR / ATM inhibitors and immune checkpoint inhibitors, which may provide additional benefit to patients in subsequent clinical trials. EXAMPLES

[0198] Example 1A: Synthesis of Drug-Linker Compounds with Glucuronide-Based Linkers: Drug-Linker Compound 1 (DL1) [ka] Synthetic route to compound 9 (also referred to herein as drug-linker compound 1 (DL1)). Chemical Preparation Protocol Step 1: Compound 1 [ka] To a stirred solution of (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromo-tetrahydro-pyran-2-carboxylic acid methyl ester (8.30 g; 20.90 mmol; 1.00 equiv.) and 4-hydroxy-3-nitro-benzaldehyde (5.24 g; 31.35 mmol; 1.50 equiv.) in acetonitrile (83.00 ml; 10.00 V) was added silver(I) oxide (9.69 g; 41.80 mmol; 2.00 equiv.). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum to give a solid. The solid was dissolved in EtOAc and washed with 10% aqueous NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to give compound 1 as a sand-colored solid. Yield: 9.0g Yield(%):89.1%

[0199] Analytical data: NMR: 1 H-NMR(400MHz,DMSO-d6):9.98(s,1H),8.46(s,1H),8.25-8.21(m,1H),7.64(d,J=11.60Hz,1H),5.94(d, J=10.00Hz,1H),5.51-5.44(m,1H),5.20-5.09(m,2H),4.80(d,J=13.20Hz,1H),3.64(s,3H),2.09(s,9H). Step 2: Compound 2 [ka]

[0200] To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 equiv.) in propan-2-ol (33.00 ml; 3.67 V) and CHCl3 (167.00 ml; 18.56 V), silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 equiv.) was added followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 equiv.). The reaction mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was quenched with chilled H2O and filtered through Celite. The filtrate was extracted with dichloromethane and dried over Na2SO4. The solvent was concentrated to give compound 2 as an off-white powder. Yield: 8.70g Yield(%): 92.4% Analysis data LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT (min): 2.05; M+H: 503.2, Purity: 96.6% Step 3: Compound 3 [ka]

[0201] To a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 equiv.) in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V) was added palladium on carbon (10% w / w) (2.50 g; 2.35 mmol; 0.14 equiv.). The reaction mixture was stirred under hydrogen atmosphere for 3 hours at room temperature. After completion, the reaction mixture was filtered through Celite. The solvent was concentrated under vacuum to give compound 3 as an off-white solid. Yield: 8.5g Yield(%): 100%

[0202] Analytical data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT (min): 1.73; M+H: 456.10, Purity: 95.1% Step 4: Compound 4 [ka]

[0203] To a stirred solution of compound 3 (7.60 g; 25.06 mmol; 1.20 equiv) in DCM (250.00 ml; 25.00 V) was added 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (15.65 g; 62.66 mmol; 3.00 equiv) at 0° C. The reaction mixture was stirred at room temperature for 16 h. After completion, the solvent was removed under reduced pressure to give the crude product. The crude product was purified by column chromatography (56% EtOAc:petroleum ether) to give the compound with 80% purity. The compound was further purified by washing with 30% EtOAc and petroleum ether to give compound 4 as a white solid. Yield: 8.5g Yield(%): 50.7%

[0204] Analytical data: LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH in H2O:ACN (95:5); B: ACN RT (min): 3.03; M+H: 735.2, Purity: 81.9% Step 5: Compound 5 [ka]

[0205] To a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 equiv.) in THF (40.00 ml; 20.00 V) at 0° C., carbonic acid bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 equiv.) and DIPEA (4.40 ml; 24.92 mmol; 10.00 equiv.) were added. The reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and petroleum ether / ethyl acetate as eluent to give compound 5 as a pale yellow solid. Yield: 2.0g Yield(%):84.6%

[0206] Analytical data:

[0207] LCMS: Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA in MilliQ water; B: ACN RT (min): 3.24; M+H: 900.20, Purity: 94.9% Step 6: Compound 6 [ka]

[0208] Compound 5 (1.369 g; 1.00 equiv.) was dissolved in N,N-dimethylformamide (15.00 ml) and exatecan mesylate (679.7 mg; 1.00 equiv.), 4-methylmorpholine for synthesis (0.422 ml; 3.00 equiv.), and 1-hydroxybenzotriazole (172.8 mg; 1.00 equiv.) were added. The reaction mixture was stirred at room temperature overnight. After the stirring time, the reaction suspension turned into a brown solution. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was purified by RP flash chromatography. The product-containing fractions were combined, concentrated under vacuum, and lyophilized overnight to give compound 6 as a yellow solid. Yield: 1.59g Yield(%): 87.5%

[0209] Analytical data:

[0210] LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100% B: 0->2.0 min; 100% B: 2.0->2.5 min RT (min): 1.95; M+H: 1196.40, Purity: 84.4% Step 7: Compound 7. [ka]

[0211] Compound 6 (1.586 g; 1.00 equiv.) was dissolved in tetrahydrofuran (50.00 ml) and a solution of LiOH (lithium hydroxide hydrate (281.77 mg; 6.00 equiv.) in water (67.100 ml)) (0.1 M) was added dropwise at 0° C. The pH value was checked during the addition. The pH should not exceed 10. The addition of the solution of LiOH was complete after 1.5 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction was quenched with citric acid solution and the pH was adjusted to 5. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC. The product-containing fractions were combined and lyophilized to give compound 7 as a dark yellow solid. Yield: 728mg Yield(%): 54.8%

[0212] Analytical data:

[0213] LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100% B: 0->2.0 min; 100% B: 2.0->2.5 min RT (min): 1.68; M+H: 1056.30, Purity: 98.5% Step 8: Compound 8 [ka]

[0214] Compound 7 (728.000 mg; 1.00 equiv.) was dissolved in N,N-dimethylformamide (20.00 ml). Piperidine (136.513 μl; 2.00 equiv.) was added and the solution was stirred at room temperature for a total of 4 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, the solvent was partially removed, and it was lyophilized overnight to give compound 8 as a yellow solid. Yield: 706mg Yield(%): 100%

[0215] Analytical data: LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100% B: 0->2.0 min; 100% B: 2.0->2.5 min RT(min): 1.22; M+H: 834.30, Purity: 97.6% Step 9: Compound 9 [ka]

[0216] To a solution of compound 8 (854 mg; 1.00 equiv.) in dimethylformamide (30.00 ml) was added N-ethyldiisopropylamine (149.234 μl; 1.00 equiv.) and 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (233.61 mg; 1.00 equiv.). The reaction mixture was stirred at room temperature for 3 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was subjected to RP flash chromatography. The product-containing fractions were combined, concentrated and lyophilized to give the desired product in 91% purity. This material was purified again by RP chromatography to give compound 9 as a yellow solid. Yield: 580mg Yield(%): 60.1%

[0217] Analytical data: LCMS:Column:Chromolith HR RP-18e (50-4.6mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow rate: 3.3 ml / min; MS: 100~2000, amu positive; 1%->100% B: 0->2.0 min; 100% B: 2.0->2.5 min

[0218] RT (min): 1.38; M+H: 985.30, purity: 90% (the other 10% of isomers can be removed by HPLC)

[0219] 1H NMR(500MHz,DMSO-d6)δ 13.10-12.44(m,1H),9.08(s,1H),8.32(t,J=5.8Hz,1H),8.16(s,1H),8.02(d,J=8.8Hz,1H),7.76(d,J=10.9Hz,1H),7.31 (s,1H),7.15-7.09(m,2H),6.98(s,2H),5.48-5.38(m,2H),5.32-5.22(m,3H),5.11-5.01(m,2H),4.87(d,J=7.6Hz,1H),3 .92-3.88(m,1H),3.89-3.84(m,2H),3.65-3.61(m,2H),3.46-3.41(m,1H),3.42-3.37(m,1H),3.38-3.31(m,1H),3.28-3. 20(m,1H),3.15-3.07(m,1H),2.48-2.44(m,2H),2.38(s,3H),2.24-2.13(m,2H),1.94-1.80(m,2H),0.88(t,J=7.3Hz,3H).

[0220] Example 1B: Synthesis of a Drug-Linker Compound with a Legumain-Cleavable Linker: Drug-Linker Compound 2 (DL2) [ka]

[0221] Step 1 {(4-[(2S)-3-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamido]propanamido]propanamido]phenyl}methyl 4-nitrophenyl carbonate (400 mg; 0.52 mmol; 1.00 equiv.) [commercially available from Levena Biopharma US] was dissolved in N,N-dimethylformamide (5.00 ml). (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 1 4.04, 13 .06, 11 .0 2°, 2 4] Tetracosa-1,6(11),12,14,16,18,20(24)-heptaene-5,9-dione; methanesulfonic acid (277.30 mg; 0.52 mmol; 1.00 equiv), N-ethyldiisopropylamine (0.27 ml; 1.57 mmol; 3.00 equiv), and 1-hydroxybenzotriazole (HOBT) (3.52 mg; 0.03 mmol; 0.05 equiv) were added. The reaction mixture was stirred at room temperature overnight. LC / MS showed complete conversion.

[0222] The crude reaction mixture was purified by preparative HPLC and lyophilized to give 365 mg (0.343 mmol) of {4-[(2S)-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamido]propanamido]propanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0], 1H-dimethyl-2-(4-(2H)-pyrrolidone)-1,3-dihydro-1H-pyrrolidone]-2,4-dihydro-1H-pyrrolidone]-2,5-dihydro-1H-pyrrolidone]-2,6-dihydro-1H-pyrrolidone]-2,7-dihydro-1H-pyrrolidone]-2,8-dihydro-1H-pyrrolidone]-2,9 ... 2 , 1 4.04, 13 .06, 11 .0 2° , 2 4]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate was obtained. LC / MS: [M+H]=1064.2 Preparative HPLC: Column: Sunfire Prep C18 OBD-75.0g (250bar) Solvent A: Water 0.1% TFA Solvent C: Solvent B: Acetonitrile 0.1% TFA [ka]

[0223] Step 2 {4-[(2S)-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamido]propanamido]propanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 1 4.04, 13 .06, 11 .0 2° , 2 4]Tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (365 mg; 0.34 mmol; 1.00 equiv.) was dissolved in N,N-(4.00 ml). Piperidine for synthesis (0.07 ml; 0.69 mmol; 2.00 equiv.) was added and the reaction solution was stirred at room temperature for 1 h.

[0224] The reaction mixture was purified by preparative HPLC to give 300 mg (0.314 mmol) of trifluoroacetic acid; {4-[(2S)-2-[(2S)-2-[(2S)-2-aminopropanamido]propanamido-3-carbamoylpropanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 1 4.04, 13 .06, 11 .0 2° , 2 4]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate was obtained. LC / MS:[M+H]:841.3 Preparative HPLC for purification: RediSep Saule:C18 130g SN:E0410A0D24BE1 Lot:262118923W Flow rate: 75ml / min [ka]

[0225] Step 3 Trifluoroacetate {4-[(2S)-2-[(2S)-2-[(2S)-2-aminopropanamido]propanamido-3-carbamoylpropanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 1 4.04, 13 .06, 11 .0 2° , 2 To a solution of 4]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (571 mg; 0.60 mmol; 1.00 equiv.) in N,N-dimethylformamide (20 ml) was added N-ethyldiisopropylamine (203 μl; 1.20 mmol; 2.00 equiv.) and N-succinimidyl 3-maleimidopropionate (162 mg; 0.60 mmol; 1.00 equiv.). The reaction mixture was then stirred for 10 min and monitored by LC / MS. The reaction mixture was purified by preparative HPLC to give 378 mg (0.35 mmol) of DL2. LC / MS:[M+H]:992.4 Preparative HPLC for purification: RediSep column: C18 86g SN:E0410A8B46130 Lot:281729189W Flow rate: 60ml / min Conditions-Volume: 264.0ml Eluent 1: A1 water 0.1% TFA Eluent 2: B1 Acetonitrile 0.1% TFA Sequence analysis: Method information: A:H2O+0.05%HCOOH|B:MeCN+0.04%HCOOH+1%H2O Temperature: 40℃ | Flow rate: 3.3ml / min | MS: 100-2000amu positive Column: Chromolith HR RP-18e 50-4.6mm 0%->100%B:0->2.0 minutes|100%B:2.0->2.5 minutes

[0226] Example 2: Immunoconjugate: Preparation of a glucuronide-based conjugate of mAb1 (called ADC1) Antibody preparation and conjugation Monoclonal antibodies (mAbs) were thawed at 2-8 °C up to 3 days prior to conjugation and stored at 2-8 °C until use. mAbs were equilibrated at room temperature on the day of conjugation before use. A mAb solution (25.3 mg / mL) in 25 mM sodium acetate pH 5.5 was pH adjusted by the addition of 4% v / v of 0.5 M Tris, 0.025 EDTA, pH 8.5. The pH adjusted mAb solution was subsequently incubated with 5 molar equivalents (relative to mAb) of TCEP for 2 h at 20 °C to reduce interchain disulfides. The solution was then incubated with 10 molar equivalents (relative to mAb) of drug-linker (DL) for 1 h at 20 °C to conjugate the DL to the antibody. The reaction was stopped by the addition of 10 molar equivalents (relative to mAb) of 20 mM N-acetylcysteine ​​(NAC) for 20 min at 20 °C. The pH was readjusted by the addition of 5% v / v 0.15 M acetic acid and vacuum filtered using a 0.22 μm PES filter unit. The reaction mixture was filtered through an Ultracel® Membrane, C Screen, 0.1 μm 2 The diafiltration was performed by TFF using a Pellicon® capsule with 10 mM histidine, 40 mM NaCl, pH 5.5 as the diafiltration buffer at 5 L / min / ml 2 A flow rate of 15 diafiltration volumes (DV) was required. After TFF, the sample was filtered again using a 0.22 μm PES filter unit and diluted to a concentration of 15 mg / ml. The final formulation of the ADC was 10 mg / ml in 10 mM histidine, 40 mM NaCl, 6% trehalose, pH 5.5. [Table 7]

[0227] Drug substance characterization Size Exclusion Chromatography (SEC) Monomer content and purity were assessed by size exclusion chromatography using a TOSOH TSKgel G3000SWXL 7.8 mm x 30 cm, 5 μm security guard column run isocratically at 0.5 ml / min for 30 min in 10% IPA, 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.95. Injection volumes ranged from 1 to 10 μl (maximum 20 μg protein). SEC Method Parameters Wavelength 280nm Column: TOSOH TSKgel G3000SWXL 7.8mm x 30cm, 5μm Mobile phase: 10% IPA, 0.2M potassium phosphate, 0.25M potassium chloride, pH 6.95 Injection volume 1-20 μL (5-20 μg protein) Column temperature: 25℃ Gradient Isocratic Elution Flow rate 0.5mL / min Run time 30 minutes Typical SEC chromatogram showing purity of stock mAb and final BDS: [ka] The BDS shown above reports 4.6% HMWS and 95.4% monomer purity.

[0228] Reverse Phase HPLC (RP HPLC) Method Reverse phase was performed on a Polymer Labs PLRP-S 2.1 mm x 50 mm, 5 μm, 1000 Å column, running at 1.0 mL / min / 80° C., with a 25 min linear gradient between 0.1% TFA 25% CH3CN (mobile phase B) and 0.1% TFA 50% CH3CN (A). Both reduced antibodies and fully conjugated ADCs were analyzed using non-reducing PLRP conditions. The methods used for sample preparation are shown in the table below: [Table 8] Typical RP-HPLC chromatogram showing separation of light and heavy chains. The chromatogram below shows the overlay of the mAb and the final BDS. [ka] For the BDS material mentioned above, a DAR of 7.8 was reported.

[0229] Hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) with gradient elution and detection by absorbance at 214 nm was used to assess the average DAR of the ADCs. The ADC molecules are listed in order of increasing DAR by decreasing salt concentration in the mobile phase consisting of 1.5 M ammonium sulfate / 20 mM sodium phosphate / pH 6.95. The column used was a Tosoh TSKgel Butyl-NPR column, 4.6 x 35 mm, 2.5 μm. Injection volumes ranged from 1 to 10 μl for antibody / conjugate samples (5 to 20 μg protein). [Table 9] Typical HIC chromatogram showing separation of mAb and ADC. The chromatogram below shows the input mAb and final BDS overlaid. [ka]

[0230] Free Drug Method Reverse phase HPLC was performed on a Phenomenex Kinetex Core Shell 2.6 μm C8 column, 100 Å, 50×4.6 mm column, operating at 2 mL / min at 60° C. with an 8 min linear gradient between 95% A (0.05% TFA / H2O) and 95% B (0.05% TFA / CH3CN). Toxin linker standards were run with NAC quenching. Data was collected at 214 nm and spectroscopically analyzed at 252, 360, and 280 nm to monitor the linker chromophore and any protein residues in the sample. Samples were deproteinized by cold methanol / salt extraction prior to HPLC analysis: 2 μl of 5M NaCl was added to 50 μl of sample, followed by 150 μl of ice-cold methanol. Samples were vortexed and incubated at −20° C. for 30 min. Precipitated proteins were pelleted by centrifugation at 15000×g, 4° C. for 30 min. 125 μl of supernatant was diluted with 125 μl of Elga 18.2 MΩ water and mixed. 100 μl of sample was injected and all data reported at 360 nm. Levels of residual toxin-related species were calculated against a standard curve of the toxin. Observed toxin amounts were reported as % mole / mole values ​​relative to the total toxin content of the sample. [ka] Crude conjugate reaction (blue), DV0 (red), DV6 (green), RP-HPLC chromatogram overlay (360 nm) of DV10 (pink) and BDS PPB-13430 (olive green).

[0231] Endotoxin Endotoxin was determined by kinetic colorimetric LAL assay using the Endosafe PTS endotoxin system (Charles River). Buffers and antibodies were diluted 10-fold in LAL reagent water. ADC was diluted 10-fold in LAL reagent water. All samples were analyzed on 0.01-1 EU / mL cartridges. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL.

[0232] GD2-ADC: In vitro and in vivo experimental results Experiment 1. Dose-response efficacy study of anti-GD2 ADC in a neuroblastoma (CHP134) xenograft model The dose-response relationship of anti-GD2 ADC molecule 1 was evaluated in the CHP134 human neuroblastoma xenograft tumor model, which expresses high levels of GD2.

[0233] Female athymic nude mice (nu / nu) bearing established CHP134 tumors (approximately 150 mm3) were treated with molecule 1 at doses ranging from 0.25 mg / kg to 1.0 mg / kg on a weekly schedule (qw) for 4 weeks via intravenous (IV) injection.

[0234] In this study, treatment was well tolerated, no toxicity was observed, and normal body weight gain was observed (Figure 1). The effect of treatment on CHP134 tumor growth and individual tumor size on days when vehicle control-treated tumors reached the study endpoint (>1,000 mm3) is shown in Figure 2. Molecule 1 administered at 0.25, 0.5, and 1.0 mg / kg on D0 (day 0), D7, D14, and D21 showed dose-dependent antitumor activity. The lowest dose tested, 0.25, showed a tumor growth inhibition (TGI) efficacy of 59%, which remained significant (p<0.01) (Figure 2). A strong activity in inducing tumor regression was observed with molecule 1 at 0.5 and 1.0 mg / kg, both doses being highly significant (p<0.0001, day 18) (Figure 2). At the end of the study (day 85), complete responses were observed in all animals (16 of 16) treated with the 0.5 mg / kg and 1.0 mg / kg doses of molecule 1 (Figure 3).

[0235] In conclusion, the results of this study demonstrated the potent antitumor activity of molecule 1, resulting in significant efficacy and tumor regression in CHP134 tumors. The minimal effective dose (MED), defined as the lowest dose to induce a >20% reduction in tumor volume from baseline (at any time point after treatment initiation), was determined to be approximately 0.5 mg / kg of molecule 1 in the CHP134 tumor model. [ka] [ka] [ka]

[0236] Experiment 2. Efficacy test of anti-GD2 ADC in osteosarcoma PDX model The efficacy of anti-GD2 ADC molecule 1 was evaluated in the GD2-expressing CTG-2735 osteosarcoma PDX model.

[0237] Female athymic nude mice (nu / nu) bearing established CTG-2735 tumors (approximately 250 mm3) were treated with a single intravenous injection of molecule 1 at a dose of 10.0 mg / kg on day 0. Group sizes for vehicle and treatment groups were 3 mice.

[0238] In this study, treatment was well tolerated, no toxicity was observed, and normal weight gain was observed (Figure 4). Treatment with 10 mg / kg of molecule 1 resulted in strong tumor growth inhibition and long-lasting tumor regression up to day 62, when the experiment was terminated (see Figures 5 and 6).

[0239] In conclusion, the results of this study demonstrated potent antitumor activity of molecule 1 in the osteosarcoma PDX model CTG-2735. [ka] [ka] [ka]

[0240] Experiment 3. Efficacy study of anti-GD2 ADC in SCLC PDX models The efficacy of anti-GD2 ADC molecule 1 was evaluated in the GD2-expressing CTG-0199 SCLC PDX model.

[0241] Female athymic nude mice (nu / nu) bearing established CTG-0199 tumors (approximately 250 mm3) were treated with a single intravenous injection of molecule 1 at a dose of 10.0 mg / kg on day 0. Group sizes for vehicle and treatment groups were 5 mice.

[0242] In this study, treatment was well tolerated, no toxicity was observed, and normal weight gain was observed (Figure 7). Treatment with 10 mg / kg of molecule 1 resulted in strong tumor growth inhibition and long-lasting tumor regression up to day 53, when the experiment was terminated (Figures 8, 9).

[0243] In conclusion, the results of this study demonstrated potent antitumor activity of molecule 1 in the SCLC PDX model CTG-0199. [ka] [ka] [ka]

[0244] Experiment 4. Efficacy study of anti-GD2 ADC and ATR inhibitor combination in a patient-derived xenograft (PDX) model of osteosarcoma (CTG-2264) The anti-tumor activity of selected ADCs of the invention was tested in the CTG-2264, an osteosarcoma PDX model.

[0245] Female athymic nude mice (nu / nu) bearing established CTG-2264 tumors (approximately 250 mm3) were treated with either a single intravenous injection of molecule 1 at a dose of 3.0 mg / kg on day 0, an ATR inhibitor at a dose of 10 mg / kg administered orally (po) daily (qd), or a combination of both treatments. Vehicle control was saline, administered on day 0 (10 mL / kg). Group size for each treatment group was 3 mice. Tumor growth inhibition (TGI) calculations were performed 23 days after treatment initiation, when the mean tumor volume in the control group had reached 1000 mm3 (see Figure 11, Figure 12).

[0246] Treatment was well tolerated with no toxicity or clinical signs and no loss in mean body weight was observed compared to initial body weight in all treatment groups (Figure 10). These ADCs alone and in combination induced tumor growth inhibition (TGI) of 65%, 67% and -72% (regression) in the CTG-2264 osteosarcoma PDX model.

[0247] In conclusion, the above combination demonstrated benefit compared with each of the two monotherapies at 10 mg / kg (p<0.0001). [ka] [ka] [ka]

[0248] Experiment 5. ADC cell killing assay against human cancer cell lines The in vitro potency of the ADC hu14.18-IgG1.4(K322A)-delK-βGluc exatecan and rituximab IgG1βGluc exatecan, the unconjugated antibody hu14.18-IgG1.4(K322A)-delK and the free payload exatecan was evaluated on the antigen-positive human tumor cell lines CHP-134 (neuroblastoma, #ACC653, DSMZ, Braunschweig, Germany), NCI-H446 (small cell lung cancer (SCLC), #HTB-171, ATCC, Manassas, VA, USA) and M21 (melanoma, Scripps Research Institute, La Jolla, CA, USA) and the antigen-negative human tumor cell line MDA-MB-468 (breast cancer, #ACC738, DSMZ, Braunschweig, Germany) using CellTiter Glo® Luminescent Cell Viability Assay (#G7573, Promega Corporation, Madison, WI, USA).

[0249] The cell lines CHP-134, NCI-H446 and MDA-MB-468 were cultured as directed by the supplier and maintained in RPMI1640 medium containing GlutaMAX™ supplement (#61870-010, purchased from Gibco™, Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate (#11360-070, Gibco™, Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) (#S0615, Sigma Aldrich, St. Louis, MO, USA). The melanoma cell line M21 was cultured in DMEM containing stable L-glutamine (#41965-039, Gibco™, Thermo Fisher Scientific) and 10% FBS.

[0250] The day before treatment, 625 cells / well (90 μl) (CHP-134, M21 or MDA-MB-468) or 1250 cells / well (90 μl) (NCI-H446) were seeded in sterile Falcon™ 96-well cell culture treated flat bottom microplates (#353219, Corning, NY, USA). After overnight incubation at 37° C. and 5% CO2 or 10% CO2, 10-fold starting concentrations of compounds and serial dilutions (1:4) were prepared using cell culture medium or RPMI 1640 medium with the supplements listed above. A total of 10 μl of compound solution was added per well (technical triplicates). Control wells were treated with the respective amounts of dimethyl sulfoxide (free payload control wells) or cell culture medium / RPMI 1640 (ADC and unconjugated antibody control wells and background wells without cells). After 6 days of incubation, 100 μl of Cell Titer-Glo® reagent was added to each well and the plate was incubated for 2 min with shaking at 300 rpm and for an additional 20 min at room temperature (protected from light). The luminescence signal was then measured in a Varioskan Flash or Varioskan Lux plate reader (Thermo Fisher Scientific) to determine cell viability. Relative light units (RLU) were processed by subtracting background and converting to % viability (RLU of untreated control cells defined as 100%) or % effect (calculated by subtracting 100% from % viability). The processed data were used to express dose response as % effect vs. concentration [M] using the equation log(inhibitor) vs. response-variable slope (4 parameters) (GraphPad Prism (version 8.2.0) for Windows, GraphPad software, La Jolla California USA, www.graphpad.com). Data are presented with error bars indicating standard deviation (SD) of technical triplicates. Several experiments were performed and the IC 50 The geometric mean of (geometric mean IC 50 [nM]) was calculated.

[0251] The cytotoxic activity of anti-GD2 hu14.18-IgG1.4(K322A)-delk-βGluc exatecan ADC (molecule 1) was determined in target-positive and target-negative human cancer cell lines. Rituximab IgG1βGluc exatecan ADC (molecule 2) with the same linker-payload and comparable DAR as well as the unconjugated antibody hu14.18-IgG1.4(K322A)-delK (molecule 3) were used as controls. Exatecan (molecule 4) was also included in the study to confirm the general sensitivity of human cancer cell lines to the free payload.

[0252] In vitro activity results are summarized in Table 1 and representative dose-response curves are shown in Figure 1-Figure 1. The anti-GD2 hu14.18-IgG1.4(K322A)-delK-βGluc exatecan ADC (molecule 1) demonstrated effective cell viability inhibition and specific sub-nanomolar or single-digit nanomolar potency against the GD2+ expressing cancer cell lines CHP-134 (Figure 1), M21 (Figure 2) and NCI-H446 (Figure 3) as shown in comparison to the non-binding control ADC (molecule 2). For the target-negative cell line MDA-MB-468 (Figure 3), which showed relatively high sensitivity to the free payload exatecan (molecule 4), no specific cytotoxic effect was observed with molecule 1 compared to the non-binding control ADC (molecule 2).

[0253] Treatment of the GD2-positive neuroblastoma cell line CHP-134 with the unconjugated antibody hu14.18-IgG1.4(K322A)-delk (molecule 3) showed cell killing activity at the highest concentration tested (Figure). This effect is in agreement with data published by Horwacik et al. (Cancer Letters, Volume 341, Issue 2, 2013, pp. 248-264), who reported a negative impact on cell viability of CHP-134 after treatment with the anti-GD2 mouse mAb 14G2a. A slight effect on cell viability after treatment with hu14.18-IgG1.4(K322A)-delK (molecule 3) was observed in the SCLC cell line NCI-H446 and the melanoma cell line M21, whereas no effect was determined in the target-negative MDA-MB-468 cell line. [Table 10] NC: Unable to calculate due to incomplete dose-response curve N: Number of experiments I C 50 : half-maximal inhibitory concentration; the concentration at which 50% of maximal inhibition was observed; the table shows the IC for the number of experiments indicated. 50 The geometric mean IC calculated from the values 50 Contains [nM]. Span (%): Highest test concentration * Percentage of total non-viable cells relative to untreated control cells; table contains geometric mean Span (%) calculated from Span (%) values ​​for the indicated number of experiments. Highest test concentration: 100 nM for molecules 1, 2, and 4; 1 μM for molecule 3 Table 1. Geometric mean IC of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan, rituximab IgG1βGluc exatecan, hu14.18-IgG1.4(K322A)-delk and payload exatecan in human cancer cell lines 50 Value [nM] and geometric mean span (%)

[0254] Figure description for Experiment 5 Figure 13 In vitro dose response curves of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan (molecule 1), control ADC rituximab IgG1βGluc exatecan (molecule 2) and payload exatecan (molecule 4) in CHP-134 cells. One representative dose response curve is shown as the mean from triplicate values ​​± SD.

[0255] Figure 14 In vitro dose response curves of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan (molecule 1), control ADC rituximab IgG1βGluc exatecan (molecule 2) and payload exatecan (molecule 4) treatment in M21 cells. One representative dose response curve is shown as the mean from triplicate values ​​± SD.

[0256] Figure 15 In vitro dose response curves of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan (molecule 1), control ADC rituximab IgG1βGluc exatecan (molecule 2) and payload exatecan (molecule 4) treatment in NCI-H446 cells. One representative dose response curve is shown as the mean from triplicate values ​​± SD.

[0257] Figure 16. In vitro dose response curves of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan (molecule 1), control ADC rituximab IgG1βGluc exatecan (molecule 2) and payload exatecan (molecule 4) in MDA-MB-468 cells. One representative dose response curve is shown as the mean from triplicate values ​​± SD.

[0258] Figure 17. In vitro dose response curves of hu14.18-IgG1.4(K322A)-delK-βGluc exatecan (molecule 1) and hu14.18-IgG1.4(K322A)-delk (molecule 3) in CHP-134 cells. Data are presented as the mean from triplicate values ​​± SD. Curves shown in the figure are from two independent experiments. [ka] [ka] [ka] [ka] [ka]

[0259] Study 6: Safety profile of ADC1: A pilot intravenous toxicity-TK study in cynomolgus monkeys To investigate the safety profile, ADC1 was administered by 30-minute intravenous infusion to cynomolgus monkeys at doses of 4 mg / kg, 8 mg / kg, 16 mg / kg and 32 mg / kg three times at weekly intervals (days 1, 8 and 15) and the animals were sacrificed (day 22) for gross and histopathological examination of a large panel of organs and tissues, including peripheral nerve tissue. Clinical signs, body weight, clinical hematological and biochemical parameters, and toxicokinetics (TK) were also included in the study. ADC1 mainly induced dose-dependent effects in the hemolymphatic and gastrointestinal systems, resembling the toxicity of exatecan (De Jager 2000, Verschraegen 2000, Rowinsky 2005). Of note, no clinical signs of pain were observed during home cage observations, or during handling of the monkeys for dose administration, or when blood samples were taken. No histopathological changes were observed in the examination of several peripheral nerve tissues (i.e., peroneal, tibial, and sciatic nerves) or central brain tissues. Adequate exposure levels of conjugated antibody in plasma were shown to be associated with very low plasma concentrations of unconjugated (free) exatecan. The absence of pain in this study is crucial, since pain or allodynia was the dose-limiting toxicity in the treatment of tumor patients with approved GD2 (IgG1) antibodies such as dinutuximab (ch14.18 / SP2 / 0, Unituxin), dinutuximab beta (ch14.18 / CHO or APN311), and naxitamab (hu3F8). Dinutuximab beta, tested in cynomolgus monkeys, showed clinical signs interpreted as acute pain confirmed by histopathological changes observed in the peripheral nervous system or in structures innervated by non-neuronal tissues (EMA / 263814 / 2017 Assessment report dinutuximab beta Apeiron). The labelling of dinutuximab beta and parent dinutuximab as well as the naxitamab antibody contains safety information regarding the issue of acute pain (treatment).Even with a GD2 antibody that mutated only the CDC effector function by the K322A mutation, pain was only partially relieved (Dobrenkov and Cheung 2014; Navid et al. 2014) and opioid use was still required (Harman et al. 2019).

[0260] Overall, the monkey safety data and mouse oncology model data with the novel ADC indicate that tumors are killed by targeted exatecan-mediated apoptosis (rather than by CDC and / or ADCC mechanisms) without the pain associated with naked anti-GD2 antibody administered alone.

[0261] Experiment 7: Evaluation of peripheral nervous system sparing ADC (molecule 1) in rats and cynomolgus monkeys To test the initial safety profile of molecule 1 in monkeys and rats expressing the same GD2 glycotope that can be targeted by the hu14.18 antibody variant used for conjugation (FDA BLA#125516, 2014; EMA / 263814 / 2017, 2017), molecule 1 showed the expected exatecan toxicity profile but did not result in any PNS damage after repeated weekly intravenous dosing in monkeys and rats. Furthermore, no signs of pain signals were observed during normal behavior of the animals in their home cages, such as locomotor activity, rearing, and exploration, or upon handling.

[0262] method The general toxicology program consisted of a pilot repeat-dose toxicity study in Wistar rats and cynomolgus monkeys with molecule 1 administered by intravenous infusion once weekly for three consecutive days (days 1, 8 and 15), including toxicokinetic assessment for exposure confirmation of the ADC (conjugated payload analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and total antibody analyzed by immunoassay) and free payload in plasma (analyzed by ultra-performance LC-MS / MS). Necropsy for gross and microscopic examination was scheduled on day 22.

[0263] Intravenous formulations: Molecule 1 (antibody manufactured by EMD Serono, Billerica, USA and conjugated by Sterling Deeside Ltd., Flintshire, United Kingdom) was formulated in a vehicle solution (control group) containing 10 mM histidine, 40 mM NaCl, 6% trehalose dihydrate, 0.05% polysorbate 20 (pH 5.5) and stored deep refrigerated (-60°C) until use. Molecule 1 stock concentration (10 mg / mL) and dilutions were verified for stability and concentration prior to administration (Merck KGaA, Darmstadt, Germany). Test substances or vehicle were administered intravenously (iv) by slow bolus injection to rats (tail vein) and by 30 min infusion to cynomolgus monkeys (peripheral vein). General parameters for toxicity evaluation. Toxicity endpoints consisted of daily clinical observations, body weights, food consumption, clinical lesions, immunophenotype, gross lesions, organ weights, and histopathology. Necropsy included examination of the carcass; external orifices; abdominal, thoracic, and cranial cavities; and organs. Tissues obtained at necropsy were preserved in 10% neutral buffered formalin, Davidson's fixative (eyes and optic nerves) or modified Davidson's (testes) fixative and processed for routine histological examination (paraffin wax-embedded tissues and HE staining). Selected organs were weighed prior to fixation. In addition, unscheduled necropsies and histology were performed on moribund animals.

[0264] B. Methodology A 3-week pilot rat toxicity and toxicokinetics study Molecule 1 was administered weekly by intravenous infusion to four groups of Crl:WI (Han) rats (5 rats / sex / group; Charles River Laboratories, Sulzfeld, Germany) at doses of 0 (vehicle control), 10, 30, and 60 mg / kg of molecule 1 for three consecutive weeks. A satellite group of three rats / sex / group was used for toxicokinetic evaluation of the three analytes. Methods: Rats were housed in groups of 2-3 rats per cage. Rats were observed daily for physical appearance, behavior, and clinical signs. Body weight was recorded before the start of treatment and then daily until the end of the study. Food intake was recorded per cage at weekly intervals until the end of the study by weighing the uningested food per cage. Hematology and clinical chemistry were performed on five rats / sex / group one week after the final dosing period (day 22). Hematological parameters were analyzed on an ADVIA 2120i autoanalyzer and clinical chemistry parameters were analyzed on an ADVIA 1800 autoanalyzer (both at Siemens Healthcare Diagnostics, GmbH). Blood samples (0.15 mL in lithium heparin tubes) from the satellite and main groups (small volume sampling) were taken for bioanalysis and toxicokinetic (TK) evaluation from all animals on days 1 and 15 through one week thereafter at the following time points: 0 hours (before dosing), and 0.5, 4, 24, 48, 96 hours (on day 1 only), and 168 hours (before the next dose) after dosing. As controls, vehicle-treated animals were sampled on days 1 and 15 (4 hours after the first and last dose).

[0265] ii. A 3-week pilot monkey toxicity and toxicokinetics study including functional assessment of cardiovascular (CV) parameters and respiratory rate Purpose-bred naive cynomolgus monkeys (Macaca fascicularis) were purchased from Envigo (Venray, The Netherlands). The monkeys were born and raised in Vietnam and were quarantined in Camarles (Camarney SLU, Spain) before being transported to the testing facility in Ivrea (Italy). The monkeys were housed in air-conditioned rooms (22 ± 2 °C) with 15-20 air changes per hour, relative humidity of 55 ± 15%, and artificial lighting with a 12-h circadian cycle from 7 am to 7 pm. The animals were group-housed in monkey enclosures fixed to the floor. The front and top walls of the enclosures were made of stainless steel bars, while the side and back walls consisted of colored plastic material. In this study, the experimental groups consisted of one animal per group and sex, but additional non-treated animals were present to ensure a social housing group of two or three subjects. The housing of non-human primates meets the Italian requirements for the welfare of laboratory animals, including an environmental enrichment program. At the start of treatment, male and female animals were approximately 3-4 years old and weighed between 3.1 and 3.8 kg. Molecule 1 was administered weekly by 30-minute intravenous infusion to one male and one female cynomolgus monkey per group for three consecutive weeks at three dose levels of the test substance (4, 8 and 16 mg / kg). One additional male monkey was infused with a fourth higher dose (32 mg / kg) to measure maximum tolerability.

[0266] Mortality and clinical signs (physical appearance, behavior, and general signs) were recorded twice daily and at the end of the infusion (up to 30 min post-dose), and body weights were recorded weekly from 1 week before the start of treatment throughout the study. Food and water were available daily (per cage). Hematology (ADVIA2120i Siemens analyzer) was performed in all monkeys during the pretreatment period and on days 3 (i.e., 48 h after the first dose), 8 (168 h before the second dose), 15 (before the third dose), 17 (i.e., 48 h after the third dose), and 22 (before sacrifice). Immunophenotyping (FACLyric Becton Dickinson flow cytometer, Becton Dickinson antibodies) was performed on the same days to determine total B cell (CD3 - , CD20 + ), whole cells (CD3 + ), helper cells (CD3 + , CD4 + ) and cytotoxic T cells (CD3 + , CD8 + ) and natural killer cells (CD3 - , CD16 + Absolute and relative numbers of IgG4 and IgG5 were measured. Clinical chemistry analyses (AU480 Beckman Coulter analyzer) were performed during the pre-dose period and on days 15 and 22.

[0267] Blood samples (0.6 mL in lithium heparin tubes) for bioanalysis and toxicokinetic (TK) evaluation were collected from each animal on days 1 and 15 up to one week thereafter at the following time points: 0 h (pre-dose), and 0.5 h (=end of infusion), 2 h, 6 h, 24 h, 48 h, 72 h, 120 h, and 168 h (pre-dose) after dosing.

[0268] iii. Integrated evaluation of electrocardiogram and arterial blood pressure As part of the pilot monkey DRF toxicity study, heart rate (HR), electrocardiogram (ECG), and arterial blood pressure (systolic BP and diastolic BP) were measured in all monkeys at baseline (pre-dose) and within 30 min of the end of infusion of the last dose (day 15). Animals were trained to record under conscious, temporarily restrained conditions while seated in a chair. Three consecutive measurements of systolic and diastolic arterial blood pressure were taken before the respiratory rate and ECG measurement session. Evaluations were made on at least 10 representative ECG complexes. Respiratory rate was measured immediately after the measurement of arterial blood pressure by counting respiratory movements over a 1-minute period. Rectal temperature was recorded in all animals twice pre-dose and on days 1, 8, and 15, at the end of infusion (within 30 min after dosing), and 24 h (± 30 min) after dosing.

[0269] Methods: ECG electrodes (disposable foam electrodes) were attached to each animal according to standard lead II. The signal was digitized (A / D converter ACQ-7700, DSI, St. Paul, Minnesota, USA) and recorded continuously once the heart rate reached a steady state using a software package (Ponemah Physiology Platform 5.20, provided by DSI). A cuff for measuring arterial blood pressure (BP) was attached to one forearm, connected to a blood pressure monitor (vital signs monitoring unit CARESCAPETM V100, GE Healthcare, Milwaukee, Wisconsin, USA). Statistical analysis was not performed due to the small number of animals per group.

[0270] Test Results: A pilot 3-week toxicity study in Wistar rats After three consecutive weekly intravenous injections of molecule 1 at three dose levels, three of 16 rats dosed at 60 mg / kg were sacrificed moribund due to clinical signs (dimpled flanks, piloerection of fur, ptosis) and a weight loss of −25% in males and −12% in females at the end of the study (day 21) compared to controls (FIG. 13). After each weekly dose, there was a transient weight loss at 60 mg / kg, the lowest value being 3-4 days after injection, with a maximum of −7% in males (maximum −27% vs. control) and −10% in females (maximum −15% vs. control) compared to the weekly weight values ​​before dosing. At 30 mg / kg, there was a −10% to −11% weight loss (compared to control) in both sexes on day 21. Over the 21-day observation period, weight gain was negligible at 60 mg / kg and nearly 50% loss at 30 mg / kg in both sexes compared to controls. Food intake was dose-dependently reduced by 33-37% at 60 mg / kg and 16-19% at 30 mg / kg.

[0271] On day 22, one week after the last dose (day 15), clinical lesion measurements showed a moderate decrease in red blood cells, hemoglobin, and hematocrit (maximum -29% for both parameters at 60 mg / kg and -9% at 30 mg / kg), and an increase in reticulocytes (maximum 90%) suggesting regeneration at the mid-dose, but no change in reticulocytes at the high-dose. At 60 mg / kg, white blood cells, lymphocytes, and eosinophils appeared to be decreased, and at 30 mg / kg, a partial decrease (eosinophils in males and lymphocytes in females). At the mid- and high-dose, platelets increased (maximum 60%), but neutrophil counts showed a tendency to decrease (not statistically significant). The day 22 chosen for hematological analysis may not have been optimal with regard to the regenerative capacity of hematopoiesis in rats. The hematological toxic effects described for exatecan in rodents (Verschraegen et al., 2000), such as reduced neutrophils, lymphocytes, and platelets, could be partially reversed 7 days after the last dose of molecule 1. Regarding clinical blood chemistry, total protein (-14%) and albumin (-12%) were reduced at 60 mg / kg only. The latter effect is most likely related to the general catabolism with no weight gain observed over a 3-week period.

[0272] Microscopic examination of organs harvested on day 22 revealed adverse findings in the hemolymphatic system (decreased cellularity and / or increased single-cell necrosis in the thymus, bone marrow (BM), and periarterial lymphoid sheath (PALS) of the spleen, and lymph nodes), gastrointestinal tract (increased single-cell necrosis in the crypt region), and reproductive organs (oocyte, granulosa cell, and seminiferous tubule degeneration), reflecting the pattern of an antimitotic cytotoxic agent. In the thymus, moderate to marked loss of the corticomedullary border was observed in the most severely affected rats. In addition, secondary findings consisting of a decrease in primary trabecular bone, reticular bone formation or fibrosis in the bone / marrow, and skin ulcers occurred, especially in one rat in the 60 mg / kg dose group. Reticular bone formation and fibrosis can be considered as an attempt to compensate for the disturbed bone formation. In four male rats in the 60 mg / kg group, skin wounds with ulcers were considered to be due to opportunistic infection (presence of cocci colonies on microscope slides) of skin changes (scratching or biting) that commonly occur under immunosuppressive conditions. The loss of germinal centers in lymph nodes of most rats observed from doses as low as 10 mg / kg and above suggested high sensitivity of this specific B cell compartment to the antimitotic activity of molecule 1 in rats. Also observed at 60 mg / kg was minimal extramedullary hematopoiesis in the liver and adrenal glands, which is considered an adaptive phenomenon due to bone marrow suppression. A moribund sacrificed female from the main 60 mg / kg group (14 days after two doses) showed multifocal moderate erosions in the cecum, which may have contributed to the deterioration of the clinical condition. Other findings in this rat were comparable to the rest of the group. Two moribund sacrificed rats from the satellite TK group (female on day 5, male on day 18) were not examined for histopathology.

[0273] Microscopic evaluation of rat dorsal root ganglia (cervical, thoracic, lumbar) and peripheral nerves including the optic, tibial, peroneal, and sciatic nerves did not reveal any abnormalities.

[0274] Toxicokinetic evaluation Total plasma exposure increased proportionally with increasing dose across the dose range tested for total antibody, conjugate (exatecan), and unconjugated (free exatecan), with accumulation of the ADC in the 10 mg / kg and 30 mg / kg treatment groups, but not in the 60 mg / kg group. Maximum plasma concentrations of unconjugated exatecan, with very low plasma levels, were observed between 0.5 and 4 hours with a half-life similar to that of the ADC (approximately 2-3 days), suggesting a rate-limiting process for its formation from the ADC. No gender differences in plasma exposure were observed.

[0275] vi. A pilot 3-week toxicity study including CV function assessment in cynomolgus monkeys Three consecutive weekly intravenous doses of molecule 1 at three dose levels were well tolerated in cynomolgus monkeys. A fourth higher dose of molecule 1 of 32 mg / kg injected into one male monkey was not tolerated after two doses (days 1 and 8) and required sacrifice on day 14. Thus, the 32 mg / kg dose was considered to exceed the MTD in this study.

[0276] Up to the MTD, monkeys showed dose-dependent gastrointestinal clinical signs (i.e., episodes of loose stools and / or diarrhea) accompanied by weight loss. At the MTD of 16 mg / kg, slight episodes of loose stools and / or diarrhea were observed in both animals and in females at 8 mg / kg. This was associated with a gradual loss of body weight over time of -11% (day 14) to -16% (day 21) in females at 16 mg / kg but not in males. Mild weight loss was observed in both sexes at 8 mg / kg and in male monkeys at 4 mg / kg. Hematology studies over time in monkeys (Figure 14) showed minimal to slight decreases in red blood cells, hemoglobin, and hematocrit at all dose levels (maximum of -27% relative to baseline values ​​in both sexes at 16 mg / kg), with limited dose-relatedness. A regenerative response (i.e., up to a 5-fold increase in reticulocyte count) was seen at 4 mg / kg and 8 mg / kg, whereas at 16 mg / kg, a moderate to severe decrease (up to -95% on day 8) was seen for the same parameters. In male monkeys, at 16 mg / kg, there was a transient decrease in neutrophil counts on days 8 and 15 (<2.0 10 3 / μL), still above the lower limit of normal (=1.10 × 10 3 / μL, average 5.77±3.57×10 3 / μL (as reported in Park et al., 2016).

[0277] At 16 mg / kg, tissue lesions were limited to lymphatic changes (hypocellularity in lymph nodes and thymus). The mild effects in the thymus in all monkeys treated with 8 mg / kg and 4 mg / kg may have been confounded by secondary stress-related effects. No histopathological changes were noted in the gastrointestinal tract.

[0278] Microscopic evaluation of peripheral nerves, including the optic, tibial, peroneal, and sciatic nerves, showed no test substance related findings at any dose tested, including in male monkeys above the MTD (necropsy on day 14).

[0279] cardiovascular function No relevant changes were measured up to the MTD for heart rate (HR), ECG (QT / HR corrected QT), systolic, diastolic and mean blood pressure, nor for respiratory rate and temperature.

[0280] Toxicokinetic evaluation Total plasma exposure increased proportionally with increasing dose across the dose range tested for total antibody, conjugate (exatecan), and unconjugated (free exatecan) without any analyte accumulation. Decreased exposure was observed after repeated dosing of 4 mg / kg in only one monkey (male), suggesting potential ADA formation in this subject. Maximum plasma concentrations of very low levels of unconjugated exatecan were usually observed after 6 hours with a half-life similar to that of the ADC (approximately 2-3 days), suggesting a rate-limiting process for its formation from the ADC. No gender differences in plasma exposure were observed.

[0281] Consideration of experimental findings Despite improved survival, acute neuropathic pain is a common and important dose-limiting adverse event (AE) associated with peripheral neuropathy observed with GD2 immunotherapy of currently marketed anti-GD2 antibodies (dinutuximab, naxitamab) in the treatment of patients suffering from neuroblastoma and osteosarcoma. In a pilot toxicity / TK study in rats and cynomolgus monkeys administered exatecan-based ADC molecule 1 intravenously once a week (3 times), no damage to the PNS was observed at day 22 and no signs of pain were observed in their behavior. Molecule 1 produced potent antitumor activity in xenograft mouse models expressing GD2, even after application of a single dose. Taken together, these preclinical data show a positive benefit-risk ratio that may improve the clinical perspective of the treatment of GD2-expressing tumors in pediatric and adult patients for current GD2 immunotherapy. Overall, no signs of abnormal defensive behavior such as hypersensitivity, anxiety, or agitation, suggestive of mechanical allodynia, were observed during handling. Also, no signs suggestive of pain were observed in home cage behavior, such as decreased locomotor activity, rearing, and exploration. In addition, trained monkeys sitting in a chair received 30 min of intravenous infusion without incident, and CV function was measured 1 h later that, although nonspecific, may respond to painful stimuli, such as tachycardia or increased blood pressure, which appeared to remain normal in the monkeys.

[0282] In contrast, 4 h / day (30 and 100 mg / m 2In a toxicity study in cynomolgus monkeys infused with dinutuximab beta at 100 mg / day, histopathological changes were observed in the peripheral nerves and splanchnic innervation of organs at day 15 (EMA / 263814 / 2017, 2017). These observations, together with clinical signs (e.g., hypoactivity) in these monkeys, were interpreted as signs most likely related to the induction of acute pain by the test substance targeting GD2 in the peripheral nervous system. After treatment of melanoma with anti-GD2 mAb (14G2a; ch14.18 is derived from this murine IgG2a isotype), some patients developed sensorimotor demyelinating polyneuropathy (Yuki et al., 1997). No toxicity studies have been conducted with naxitamab (FDA BLA#761171, 2020) or dinutuximab (Unituxin®, FDA BLA#125516, 2014) in monkeys or other non-rodent species.

[0283] In the DRF toxicity study, no microscopic changes were observed in several tested peripheral nerves (sciatic, tibial, peroneal, and optic nerves in monkeys and rats, as well as different levels of DRG in rats) after repeated application of molecule 1, but adequate exposure to the ADC in plasma was confirmed. Clinical signs in monkeys were limited to sporadic occurrences of mild loose stools and diarrhea only at the MTD of 16 mg / kg, as well as weight loss in females (-16%) (not observed in males). A dose-dependent decrease in weight gain and a slight decrease in food consumption were observed, mainly in rats. At 60 mg / kg, 3 of 16 rats were in poor condition and did not gain weight, so the higher dose in this DRF rat study was considered to exceed the MTD. Clinical and histopathological examinations showed that monkeys and rats exhibited hematolymphatic and gastrointestinal effects similar to exatecan toxicity (Verschraegen et al., 2000; Rowinsky 2005) due to relatively low plasma levels of unconjugated exatecan released from the ADC over a one-week period. Neutropenia, along with other hematologic toxic effects (such as anemia, lymphopenia, and thrombocytopenia) and gastrointestinal effects, is considered the primary dose-limiting toxicity with exatecan mesylate as observed in oncology patients and animals.

[0284] As detailed in Experiment 7, no signs of immunogenicity were observed in rats and monkeys after repeated dosing of molecule 1 over a period of 3 weeks (except for one low-dose monkey), as judged by the TK profile showing normal dose proportionality and regular TK curves, and the absence of unusual toxicity. The lack of immunogenicity is likely explained by the strong immunosuppressive effects induced by molecule 1 (e.g., absence of germinal centers, the major B-cell compartment). Other non-hematologic toxic effects observed with molecule 1, such as antiproliferative changes in reproductive organs / tissues in female and male rats, may be due to the antimitotic effects of exatecan (Verschraegen et al., 2000, Rowinsky 2005).

[0285] By modifying the concept of tumor killing by replacing it with targeted delivery of a non-neurotoxic payload and removing any antibody Fc-effector function, PNS damage was prevented in two test animal species. The high advantage of this consistent result is that molecule 1 may at least eliminate or reduce potential harm to the patient's PNS.

[0286] In some embodiments of the present invention, treatment methods describe the PNS-sparing activity of molecule 1 such that ocular neuropathy (eg, mydriasis, blurred vision, anisocoria, or photophobia) may be prevented.

Claims

1. An antibody-drug conjugate ("ADC") comprising a growth inhibitor and / or antiproliferative agent conjugated to an antibody via a linker, wherein the antibody comprises the light chain variable region of the anti-GD2 antibody of SEQ ID NO: 1, the heavy chain variable region of the anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a single mutation that reduces complement binding compared to antibody-dependent cell-mediated cytotoxicity, wherein the single mutation results in an absolute decrease in complement binding.

2. The antibody according to claim 1, wherein the single mutation eliminates complement binding.

3. The antibody according to claim 1, wherein the Fc region is derived from IgG.

4. The antibody according to claim 3, wherein the IgG is IgG1.

5. The antibody according to claim 1, further comprising a CH1 domain.

6. The antibody according to claim 5, wherein the Fc region is derived from IgG.

7. The antibody according to claim 5, wherein the IgG is IgG1.

8. The antibody according to claim 1, further comprising a CL domain.

9. The antibody according to claim 8, further comprising a CH1 domain.

10. The linker according to claim 1, wherein the linker is cleavable by glucuronidase.

11. The linker according to claim 1, wherein the linker is cut by Regmine.

12. The growth inhibitor according to claim 1, wherein the growth inhibitor is exatecan.

13. The ADC according to claim 1, having an average drug-antibody ratio in the range of 1 to 10.

14. The ADC according to claim 1, having an average drug-antibody ratio in the range of 2 to 4.

15. The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence shown in SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence shown in SEQ ID NO:

1.

16. A pharmaceutical composition comprising the ADC described in claim 1 and a pharmaceutically acceptable excipient.

17. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 16.

18. An antibody-drug conjugate ("ADC") comprising a growth inhibitor and / or antiproliferative agent conjugated to an anti-GD2 antibody via a linker, wherein the anti-GD2 antibody comprises the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 1, the linker is β-glucuronide, and the growth inhibitor is exatecan.

19. The ADC according to claim 18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence shown in SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence shown in SEQ ID NO:

1.

20. A pharmaceutical composition comprising the ADC described in claim 18 and a pharmaceutically acceptable excipient.

21. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 20.

22. The method according to claim 21, wherein the administration of the pharmaceutical composition is associated with a reduction in peripheral neuropathy associated with the administration of other ADCs approved for use in humans.

23. An immunoconjugate comprising an anti-GD2 antibody covalently linked to exatecan via a linker, wherein the anti-GD2 antibody comprises the amino acid sequence of SEQ ID NO: 4 (heavy chain sequence) and the amino acid sequence of SEQ ID NO: 1 (light chain sequence), and the conjugate has the following formula: 【Chemistry 1】 It has, An immunoconjugate in which S is a sulfur atom of the antibody, n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and n is between 7.5 and 8.0.

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