Anti-gd2 antibodies and immunoconjugates for the treatment of gd2-positive cancers

Abstract

This invention provides antibodies that bind to the human GD2 protein, as well as isolated nucleic acids and host cells comprising sequences encoding said antibodies. This invention also provides immunoconjugates comprising said antibodies linked to growth inhibitors, and pharmaceutical compositions comprising the antibodies or immunoconjugates of this invention. This invention further provides the use of the antibodies, immunoconjugates, and pharmaceutical compositions of this invention for cancer treatment or diagnostic purposes.

Description

Technical Field

[0001] This invention relates to antibodies binding to human GD2 (disialylganglioside GD2), and to isolated nucleic acids and host cells comprising sequences encoding said antibodies. The invention also relates to immunoconjugates comprising said antibodies linked to growth inhibitors, and pharmaceutical compositions comprising the immunoconjugates of the invention. Furthermore, the invention relates to the use of the antibodies, immunoconjugates, and pharmaceutical compositions of the invention for cancer treatment and / or diagnostic purposes. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of therapeutic agents that combine the specificity of monoclonal antibodies (mAbs) with the potency of cytotoxic molecules. The use of ADCs confers cancer-killing activity to antibodies through the conjugated cytotoxic agent, while target-specific delivery reduces systemic toxicity caused by exposure to the free cytotoxic agent. Currently, a total of twelve ADCs have been approved by the FDA for the treatment of human cancers, most of which were approved in the past two to three years. The first two approved ADCs were: ADCETRIS® (brentuximab vedotin or SGN-35), an anti-CD30 antibody conjugated to the cytotoxic agent MMAE, designed for the treatment of CD30-positive relapsed lymphoma, and KADCYLA® (T-DM1), an anti-HER2 antibody conjugated to the cytotoxic agent DM1, designed for the treatment of HER2-positive metastatic breast cancer. At least eight more drugs have subsequently been approved from these initial approvals.

[0003] Connector technology significantly impacts the potency, specificity, and safety of ADCs. Enzyme-cleavable connectors leverage the differential activity of intracellular and extracellular proteases to control drug release. Drugs can be conjugated to antibodies via a variety of different connectors and are specifically cleaved only by lysosomal proteases, which are present intracellularly and at elevated levels in certain tumor types (Koblinsk et al., 2000). This ensures the stability of the connector in the bloodstream, limiting damage to healthy tissues. However, the increased hydrophobicity associated with some enzyme-instable connectors can lead to ADC aggregation, particularly of strongly hydrophobic drugs. Therefore, there is a need for connectors that provide serum stability as well as increased solubility, allowing for efficient conjugation and intracellular delivery of hydrophobic drugs.

[0004] The literature provides examples of anti-GD2 monoclonal antibodies used in the treatment of neuroblastoma and other tumor indications. Currently, three naked anti-GD2 antibody drugs are approved for human use: naxitamab (Danyelza®) and dinutuximab (Unituxin®) in the United States, and dinutuximab β (Qarziba®) in Europe. While dinutuximab and dinutuximab β are mouse-human chimeric antibodies (ch14.18) produced in mouse myeloma cell lines designated SP2 / 0 and CHO (Chinese hamster ovary) cells, respectively, naxitamab is a humanized antibody (hu3F8) produced from CHO cells.

[0005] Approximately 12% of all pediatric cancer patients die from neuroblastoma, the most common extracranial solid tumor in childhood. Most patients are diagnosed with high-risk neuroblastoma, which has a 50% mortality rate. Treatment for high-risk neuroblastoma consists of multimodal therapy, which is associated with severe short- and long-term toxicity. Relapse rates are high, and there are limited options for further treatment. Intensification of conventional therapy has not improved outcomes and is even associated with increased toxicity.

[0006] As noted above, dartuximab, an antibody targeting ganglioside GD2, has been FDA-approved for the treatment of neuroblastoma, a carbohydrate-containing sphingolipid antigen consistently expressed in neuroblastoma, neuroectodermal cancer types, and neural tissue. Administration of this antibody in combination with cytokines and differentiation factors has improved patient outcomes and demonstrated the sensitivity of neuroblastoma 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: pp. 349–62). Dartuximab has improved event-free survival compared to previous standard chemotherapy. However, the positive clinical outcomes observed with administration of anti-GD2 antibodies alone are sometimes associated with serious toxicity. While numerous toxicities have been documented (e.g., tachycardia, hypertension, hypotension, fever, and urticaria), the most debilitating toxicity to date is neuropathic pain. Indeed, despite co-administration of potent analgesics (including opioids), this neuropathic pain often limits the dosage and, consequently, the efficacy of anti-GD2 antibodies. This neuropathic pain is likely mediated by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] Furthermore, progress has been made in introducing disialotetrahexosylganglioside (GD2) immunotherapy for patients with high-risk neuroblastoma (12 months or older) or refractory / recurrent neuroblastoma (Ahmed et al., 2014; Nazha et al., 2020). However, neuropathic pain is a common and critical dose-limiting adverse event, hindering the full therapeutic potential of currently marketed GD2 antibodies (datuximab, nalcitrumab) for these patients. It appears that the binding of anti-GD2 Abs to GD2 expressed on peripheral somatosensory and visceral nerves mediates severe acute and / or atypical pain in various body regions and is associated with peripheral nerve injury in patients (Yuki et al., 1997) and monkeys (EMA / 263814 / 2017, 2017). Although GD2 is expressed at relatively low levels in peripheral nerves (Slart et al., 1997; Lammie et al., 1993; Yuki et al., 1997; Vriesendorp et al., 1997), it is highly likely that the Fc effector functions of Ab, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (which should also kill tumor cells), trigger an immune attack on peripheral nerves via 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 monomethylaurestatin E (MMAE), a potent anticancer microtubule-targeting agent (MTA), used as the payload in three approved MMAE-containing ADCs (and several others currently in clinical development) to treat different types of cancer. MMAE-ADCs frequently induce peripheral neuropathy, a frequent adverse event that leads to dose reduction or discontinuation of many MMAE-ADCs and subsequent clinical termination. MMAE-ADC-induced peripheral neuropathy is attributed to the nonspecific uptake of the ADC in the peripheral nerves and the release of MMAE, which disrupts microtubules (MTs) and leads to neurodegeneration.

[0009] Gangliosides are composed of ceramides bound to sialic acid-containing glycan chains and are located at the plasma membrane. Gangliosides are produced through a sequential process of glycosylation and sialylation. This compositional diversity is reflected in the differences in expression patterns and functions among various gangliosides. Ganglioside GD2 follows a basic structure containing three carbohydrate residues and two sialic acids, designating different subspecies. GD2 expression, usually confined to limited tissues, is frequently altered in various neuroectodermal-derived cancers. When overexpressed in cancer, GD2 has been shown to enhance cell survival and invasion. Furthermore, antibody binding leads to immune-independent cell death mechanisms. Additionally, GD2 contributes to T cell dysfunction and acts as an immune checkpoint. Cytotoxic effectors (in one instance, drug conjugates) can target solid tumor cancers by targeting receptors against anti-GD2 antibodies.

[0010] Glycosphingolipid GD2 is a cell membrane component of a limited number of normal cells, including nerve cells, melanocytes, or lymphocytes. However, in cancers (e.g., neuroblastoma, osteosarcoma, glioma, melanoma, and soft tissue sarcoma), GD2 is found to have high antigenic levels on the cell membrane. This expression pattern makes GD2 an attractive target for therapeutic antibodies, such as dartuximab (CH14.18) and nalcitumab, which are already approved for neuroblastoma.

[0011] Therefore, what is needed is an anti-GD2 ADC, whose antibody preferably has reduced CDC and ADCC, conjugated with a non-neurotoxic cytotoxic payload (partly due to its chemical configuration and formulation), which can more selectively deliver its cytotoxic payload to malignant cells without triggering toxicity and, in particular, peripheral neuropathy that otherwise limits dosing and efficacy. Summary of the Invention

[0012] The humanized anti-GD2 antibody hu14.18 binds to disialotetrahexosylganglioside GD2, which is found on the cell surface of tumors (including neuroblastoma, melanoma, sarcoma, and others) and in normal tissues primarily confined to the central and peripheral nervous systems in humans. Expression of disialotetrahexosylganglioside GD2 in normal tissues is largely limited 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 the function of antibody effectors affecting peripheral nerves (e.g., complement fixation and complement-dependent cytotoxicity (CDC)). Previously, an antibody against hu14.18, designated "hu14.18-IgG1(K322A)" (described by reference in U.S. Patent 8,835,606 B2, incorporated herein by reference), was developed with a point mutation K322A (Kabat EU index number) in the constant domain of immunoglobulin IgG1. This K322A point mutation in the hu14.18 antibody is designed to prevent activation of complement cascade and CDC effector functions while maintaining the potential for antibody-dependent cell-mediated cytotoxicity (ADCC) effector functions. Additionally, hu14.18-IgG1(K322A) with low fucosylation was produced to enhance ADCC activity (as described in the entire amino acid and nucleic acid sequence of U.S. Patent 8,835,606 B2, incorporated herein by reference). ADCC-enhanced hu14.18-IgG1(K322A) can 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 has not yet been approved. A chimeric anti-GD2 antibody with a wild-type IgG1 isotype (dartuximab) has been approved for a limited cohort of pediatric neuroblastoma patients who are known to experience neuropathic pain associated with this treatment and who may request co-administration with analgesics.

[0013] In some embodiments, the present invention describes anti-GD2 hu14.18 antibodies as ADCs, wherein both ADCC and CDC antibody effector functions are reduced or absent, thereby providing a larger therapeutic window to address unmet medical needs. Designing hu14.18 antibodies without significant ADCC or CDC effector functions can reduce the risk of neuropathic pain induced by administration of anti-GD2 hu14.18 antibodies, and the conjugation of drugs to antibodies provides a different mechanism of action for the elimination of tumor cells by such anti-GD2-ADCs. The combination of anti-GD2 hu14.18 lacking significant ADCC and CDC effector functions (whose delivery is a potent conjugated drug as an ADC) improves existing treatments and has the potential to meet patients' (to date) unmet medical needs. In selected embodiments, the selected cytotoxic drug should not have neurotoxic properties.

[0014] The binding of an antibody to a specific antigen target is a property of the antigen-binding fragment (Fab) domain, which includes the amino acid sequence of a specific light chain variable region and a heavy chain variable region complementation-determining region (CDR). The effector function of an antibody and its binding to the Fcγ receptor (FcγR) and neonatal Fc receptor (FcRn) are properties of the immunoglobulin (Ig) constant domains. Different immunoglobulin constant domain genes exist, and these constant domain genes, fused 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 with different constant domain isotypes to produce antibodies with the same antigen target binding specificity but different effector functional properties. The ability of an antibody to mediate the activation of components of the immune system is called the antibody's "effector function," and important effector functions are mediated through interactions with FcγR on immune cells and complement proteins such as complement component 1q in blood and extracellular fluid. The binding of (C1q) is used to mediate this process. Depending on the isotype, antibodies associate specific antigenic targets on cells with the immune system and can induce targeted attacks on target cells by immune functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). After the antibody has been internalized into the cell, the binding of the Ig constant domain to FcRn plays an important role in the in vivo half-life of the antibody through increased recycling. Numerous investigations have been conducted testing the effects of specific amino acid residues in the antibody constant domain on antibody effector function and the binding of the antibody to FcγR and complement proteins. Some changes in the amino acid sequence of the human Ig constant domain can be used to generate recombinant antibodies with altered properties regarding antibody effector function and antibody binding to FcγR and complement proteins. In the selected embodiment, the present invention provides an ADC protein that binds to the target GD2 to deliver ADC molecules to cells without significant antibody effector function.

[0015] In some embodiments of the invention, the production and use of an anti-GD2 hu14.18-IgG1.4(K322A)-delK antibody are described, said antibody being conjugated to a small molecule toxicity payload from the Exatecan topoisomerase I inhibitor class for the treatment of various oncological indications, such as sarcoma, neuroblastoma, and SCLC. This novel ADC provides a preferred therapeutic window compared to other GD2 antibody therapies already in clinical use.

[0016] In a preferred embodiment of the invention, the Fc partial effector is functionally removed from the ADC, and conversely, the non-neurotoxic payload eczema is used to kill tumor cells, resulting in the absence of PNS damage and significant clinical pain signals in rats and monkeys after repeated infusions of molecule 1.

[0017] In selected embodiments of the invention, an anti-GD2 ADC [i.e., an anti-GD2 hu14.18-IgG1.4(K322A)-delK antibody, designated molecule 1, conjugated with a small molecule toxic payload from eczematopoisomerase class I via a linker] is used in novel therapeutic agents for treating cancers with high levels of GD2 expression on their cell surface. In some embodiments, the payload linker comprises the TOP1i payload eczematopois and a cleavable β-glucuronide linker structure, which has demonstrated higher potency, better bystander effect, and improved activity in multidrug-resistant cancer cells compared to other TOP1i warheads. The antitumor effects of approved anti-GD2 antibodies to date are associated with antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Molecule 1 utilizes high antigen density for direct tumor cytotoxicity via the payload. Because ADCC and CDC effector functions cause side effects similar to those of other GD2 antibodies approved to date, molecule 1 antibody is partially modified with mutations in the Fc region to reduce or eliminate these Fc-mediated functions. In the GD2-positive neuroblastoma cell line CHP134, molecule 1 exhibits sub-nanomolar IC50 cytotoxicity. At doses ranging from 3.0 to 10.0 mg / kg, molecule 1 demonstrated potent in vivo antitumor activity with tumor regression in CHP134 xenograft models and in patient-derived xenografts from neuroblastoma, osteosarcoma, or glioma. Molecule 1 showed favorable pharmacokinetics in monkeys, indicating a stable linker-payload and a favorable safety profile.

[0018] In one embodiment, the present invention describes a pharmaceutical composition for treating solid tumor cancers, wherein the solid tumor cancers exhibit high levels of cell surface GD2 expression, the pharmaceutical composition comprising an antibody-drug conjugate (ADC), wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked 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 mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation. In one embodiment, the growth inhibitor in the above pharmaceutical composition is eczema. In another embodiment, the above solid tumor cancers are selected from osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma. Detailed Implementation

[0019] definition “GD2” is a disialic acid ganglioside expressed in neuroectodermal tumors (including human neuroblastoma and melanoma), exhibiting highly restricted expression in normal tissues, primarily confined to the central and peripheral nervous systems in humans. GD2 is defined by the following chemical structure: It corresponds to the following 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)oxacyclohexane-2-yl]oxy-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2 ... )-6-[(E)-3-hydroxy-2-(octadecylamino)octadec-4-enoxy]oxacyclohexane-3-yl]oxy-3-hydroxy-6-(hydroxymethyl)oxacyclohexane-4-yl]oxy-3-amino-6-carboxy-4-hydroxyoxacyclohexane-2-yl]-2,3-dihydroxypropoxy]-5-amino-4-hydroxy-6-(1,2,3-trihydroxypropyl)oxacyclohexane-2-carboxylic acid.

[0020] As used herein, the designation "M4344" refers to a competitive inhibitor of adenosine triphosphate (ATP) having the following chemical structure: A "domain" or "region" can be any region of a protein, is generally defined based on sequence homology, and is often associated with a specific structural or functional entity. Members of the GD2 family are known to consist of Ig-like domains. The term "domain" is used in this document to specify an individual Ig-like domain, such as "N domain," or a contiguous group of domains, such as "A2-B2 domains."

[0021] A "coding sequence" or 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; that is, the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. The coding sequence of a protein may include a start codon (typically ATG) and a stop codon.

[0022] As used herein, references to a specific protein (e.g., an antibody) may include polypeptides having a natural amino acid sequence, as well as variant and modified forms, regardless of their origin or mode of preparation. A protein having a natural amino acid sequence is a protein having the same amino acid sequence as that derived from nature. Such naturally occurring sequence proteins can be isolated from nature or prepared using standard recombinant and / or synthetic methods. Natural sequence protein specificity encompasses naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternative splicing forms), naturally occurring allelic variants, and forms including those with post-translational modifications. Natural sequence proteins include proteins carrying post-translational modifications (e.g., glycosylation, phosphorylation, or other modifications to some amino acid residues).

[0023] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence comprising all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but not translated into amino acid sequences. Other genes can act as regulators of structural genes or regulators of DNA transcription. In particular, the term gene can refer to a genomic sequence that encodes a protein, i.e., a sequence that contains regulatory sequences, promoters, introns, and exons.

[0024] In this paper, a sequence that “has at least 85% identity” with a reference sequence is one that has 85% or higher sequence identity with the reference sequence over its entire length, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percentage of “sequence identity” can therefore be determined by comparing two such sequences over their entire length using the global pairwise alignment of Needleman and Wunsch’s algorithm (J. Mol. Biol. 48:443 (1970)), for example using the Needle (EMBOSS) procedure with a BLOSUM62 matrix and the following parameters: vacancy opening = 10, vacancy extension = 0.5, terminal vacancy penalty = false, terminal vacancy opening = 10, terminal vacancy extension = 0.5 (which is the standard setting).

[0025] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a side chain that has similar chemical properties (e.g., charge, size, or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. Examples of amino acid groups with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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 the size of the amino acids.

[0026] Antibodies (also known as immunoglobulins) can be, for example, natural or conventional types of antibodies, in which two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two classes of light chains: λ (I) and κ (k). There are five main classes (or isotypes) of heavy chains that determine various aspects of the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains different sequence domains (or regions). The light chain of a typical IgG antibody typically includes two regions: a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody typically includes four regions: a variable region (VH) and a constant region (CH), the latter consisting 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 both the light and heavy chains can confer important biological properties such as antibody chain binding, secretion, transplacental migration, complement fixation, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of the antibody and consists of a variable portion of a light chain and a heavy chain.

[0027] The specificity of an antibody lies in the structural complementarity between its binding site and the antigenic determinant. The antibody binding site is composed primarily of residues from the so-called hypervariable region or complementarity-determining region (CDR). Therefore, the complementarity-determining region (CDR) refers to the amino acid sequence that collectively defines the binding affinity and specificity of the antibody's Fv region. The light chain (L) and heavy chain (H) of an antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the antigen-binding site of a conventional antibody comprises six CDRs, which include sets of CDRs from the variable regions of both the heavy and light chains.

[0028] The “frame region” (FR) refers to the amino acid sequence between the CDRs, specifically those portions of the variable regions of the immunoglobulin light and heavy chains, which are relatively conserved across different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, the “human frame region” is a frame region that is substantially identical (approximately 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the frame region of naturally occurring human antibodies.

[0029] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule having a single amino acid sequence that targets a specific antigen, and should not be construed as requiring antibody production via any particular method. Monoclonal antibodies can be produced, for example, from a single clone of B cells or hybridomas, but can also be recombinant, for example, produced via methods involving genetic or protein engineering.

[0030] The term "chimeric antibody" refers to a modified antibody, in its broadest sense, containing 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 VH and VL of an antibody derived from a non-human animal that binds to CH and CL of another antibody; in some embodiments, said other antibody is a human antibody. Any animal can be used as a non-human animal, such as a mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also indicate multispecific antibodies that are specific for at least two different antigens.

[0031] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified to replace certain amino acids in framework regions such as VH and VL in order to avoid or minimize the immune response in humans. The constant regions of humanized antibodies are typically the human CH and CL regions.

[0032] An antibody (such as a conventional antibody) "fragment" contains a portion of a complete antibody, such as IgG, specifically the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, biantibodies, and bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single-domain antibodies, such as heavy chain antibodies or VHH.

[0033] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, wherein approximately half of the N-terminal side of the heavy chain and the entire light chain are linked together by disulfide bonds. It is typically obtained as a fragment by treating IgG with the protease papain.

[0034] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity, slightly larger than two identical Fab fragments bound via disulfide bonds in the hinge region. It is typically obtained from fragments by treating IgG with the protease pepsin.

[0035] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, obtained by cleaving the disulfide bonds in the hinge region of F(ab')2.

[0036] A single-chain Fv ("scFv") is a covalently linked VH::VL heterodimer, typically expressed by a gene fusion comprising VH and VL encoding genes linked via peptide-coding linkers. The human scFv fragments of this invention comprise, for example, CDRs that maintain an appropriate conformation using recombination techniques. Bivalent and multivalent antibody fragments can be spontaneously formed by the binding of monovalent scFvs, or generated by coupling monovalent scFvs, such as divalent sc(Fv)2, via peptide linkers. "dsFv" refers to a VH::VL heterodimer stabilized by disulfide bonds. "(dsFv)2" indicates two dsFvs coupled via peptide linkers.

[0037] An "antibody-drug conjugate" or "ADC" is an antibody conjugated to one or more cytotoxic agents via a linker. This antibody is typically a monoclonal antibody specific to an antigen. In some preferred embodiments of the invention, the ADC is designed as a targeted therapy for treating cancer. Unlike chemotherapy alone, these preferred embodiments combine the targeting ability of a monoclonal antibody with the cancer-killing ability of a cytotoxic drug, and can differentiate between healthy and malignant tissues.

[0038] As used in this article, "molecule 1" (also referred to as "M3554" in this article) refers to... Figure 26 The ADC shown Wherein: the antibody binds to GD2, the antibody contains 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 eczema.

[0039] The term "hybridoma" refers to cells obtained by fusing B cells prepared by immunizing non-human mammals with antigens with myeloma cells derived from mice, etc., said cells producing desired monoclonal antibodies with antigen specificity.

[0040] In some instances, the designation "M3554" is synonymous with molecule 1.

[0041] When referring to polypeptides (e.g., antibodies) or nucleotide sequences, “purified” or “isolated” means that the indicated molecule is present in the absence of other biomolecules of the same type. As used herein, the term “purified” means the presence of at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of the same type of biomolecule. An “isolated” nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or portions that do not harmfully affect the essential properties of its composition.

[0042] As used herein, the term "subject" refers to a mammal, such as a rodent, feline, canine, primate, or human. In embodiments of the invention, the subject (or patient) is a human.

[0043] As used in this article, the term "peripheral neuropathy" refers to damage to nerves (peripheral nerves) located outside the brain and spinal cord, which clinically manifest as weakness, numbness, and / or pain.

[0044] As used herein, “neuropathic pain” refers to pain caused by damage or injury to nerves that transmit information from the skin, muscles, and other parts of the body between the brain and spinal cord. This pain is clinically manifested as a burning sensation, and the affected area is often sensitive to touch.

[0045] Method for producing the antibody of the present invention The antibodies of the present invention can be produced by any technique known in the art, either alone or in combination, such as, but not limited to, any chemical, biological, genetic or enzymatic techniques.

[0046] Knowing the amino acid sequence of the desired antibody, those skilled in the art can readily produce the antibody or immunoglobulin chain using standard techniques for peptide production. For example, they can be synthesized using well-known solid-phase methods, using commercially available peptide synthesis instruments (such as those manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies and immunoglobulin chains of the present invention can be produced using recombinant DNA techniques, as well known in the art. For example, after incorporating a DNA sequence encoding the desired peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host expressing the desired peptide, these peptides (e.g., antibodies) can be obtained as DNA expression products, which can then be subsequently isolated from the host using well-known techniques.

[0047] The amino acid sequence of the antibody chain is used to produce a protein precursor for the anti-GD2-ADC molecule. 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 with G446 (Kabat EU index number) or K447 (Kabat EU index number) as shown. Changes in the constant domains compared to the wild-type human IgG1 sequence are underlined.

[0048] The mature light chain amino acid sequence of hu14.18: hu14.18-IgG1-delK mature heavy chain amino acid sequence: hu14.18-IgG1.4-delK mature heavy chain amino acid sequence: hu14.18-IgG1.4(K322A)-delK heavy chain amino acid sequence exist Figure 27 The document provides a flowchart for characterizing candidate proteins for testing anti-GD2 antibody precursor proteins.

[0049] The present invention further relates to a method for producing the antibody of the present invention, the method comprising the steps of: (i) culturing a host cell transformed according to the present invention; (ii) expressing an antibody; and (iii) recovering the expressed antibody.

[0050] The antibodies of the present invention can be appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0051] 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 previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL, and expressing the encoding sequences by introducing the expression vector into animal cells.

[0052] For the CH domain of a human chimeric antibody, any region belonging to the human immunoglobulin heavy chain, such as those of the IgG class, is suitable, and any subclass of the IgG class, such as IgG1, IgG2, IgG3, and IgG4, can be used. Similarly, for the CL domain of a human chimeric antibody, any region belonging to the human immunoglobulin light chain can be used, and regions of the κ or λ class can be used.

[0053] Methods for producing humanized or chimeric antibodies may involve conventional recombinant DNA and gene transfection techniques well known in the art (see, for example, Morrison SL. et al. (1984) and patent documents US 5,202,238; and US 5,204,244).

[0054] 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 a variety of techniques known in the art, including, for example, those disclosed in application WO2009 / 032661, CDR transplantation (EP 239,400; PCT disclosure WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), inlay or surface reshaping (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain tampering (U.S. Patent No. 5,565,332). Common recombinant DNA techniques for preparing such antibodies are also known (see European patent application EP 125023 and international patent application WO 96 / 02576).

[0055] The Fab of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with a protease, such as papain. Alternatively, the Fab can be produced by inserting the DNA sequence of the two strands of the Fab encoding the antibody into a vector for prokaryotic or eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as the case may be) to express the Fab.

[0056] The F(ab')2 of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with the protease pepsin. Alternatively, F(ab')2 can be produced by binding Fab' as described below via a thioether bond or a disulfide bond.

[0057] 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, Fab' can be produced by inserting the DNA sequence of the Fab' chain encoding the antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as the case may be) to perform its expression.

[0058] The scFv of the present invention can be produced by: obtaining the sequence of the CDR or VH and VL domains as previously described for the antibody 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 prokaryotic or eukaryotic cells (as the case may be) to express the scFv. To generate humanized scFv fragments, a well-known technique known as CDR transplantation can be used, which involves selecting the complementarity-determining region (CDR) according to the present invention and transplanting it into a human scFv fragment framework having a known three-dimensional structure (see, for example, WO98 / 45322; WO 87 / 02671; US ​​5,859,205; US 5,585,089; US 4,816,567; EP0173494).

[0059] Modified anti-GD2 antibody Amino acid sequence modifications of the antibodies described herein have been considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0060] The structure of the antibodies of the present invention and the DNA sequence encoding them can be modified and altered, and functional antibodies or peptides with the desired properties can still be obtained.

[0061] When altering the amino acid sequence of a polypeptide, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index for protein-protein interactions and biological functions is generally understood in the art. It is generally accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interactions between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid has been assigned a hydrophilic index based on its hydrophobic and charge properties. 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).

[0062] A further aspect of the invention also covers functionally conserved variants of the polypeptides of the invention.

[0063] For example, certain amino acids can be substituted by other amino acids in the protein structure without a significant loss of activity. Since the interacting abilities and properties of proteins limit their biological activity, certain amino acid substitutions can be made in the protein sequence, and naturally, in its encoding DNA sequence, while still obtaining a protein with similar properties. Therefore, it is contemplated that various alterations can be made in the antibody sequence of the present invention or in the corresponding DNA sequence encoding the polypeptide without a significant loss of its biological activity.

[0064] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophilicity indices or scores, and proteins with similar biological activities can still be obtained, i.e., proteins that are still biologically functionally equivalent. Using well-established techniques, such as alanine scanning, it is also possible to identify, in the antibodies or peptides of the present invention, all amino acids that can be substituted without a significant loss of antigen binding. Such residues can be considered neutral because they are not involved in antigen binding or maintaining the antibody's structure. One or more of these neutral positions can be substituted with alanine or another amino acid without altering the key properties of the antibodies or peptides of the present invention.

[0065] A neutral position can be considered a position where any amino acid substitution can be incorporated. In fact, in the principle of alanine scanning, alanine is chosen because this residue does not carry a specific structural or chemical signature. It is generally accepted that if alanine can substitute for a particular amino acid without altering the properties of the protein, then many other (if not all) amino acid substitutions are likely to be neutral as well. Conversely, in cases where alanine is a wild-type amino acid, if a particular substitution can be neutral, then it is likely that other substitutions will also be neutral.

[0066] As outlined above, amino acid substitutions are generally based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account any of the foregoing properties are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0067] It may also be desirable to modify the antibodies of the present invention in terms of effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or, for example, to alter binding to Fc receptors. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in that region. The resulting homodimeric antibody may thus have improved internalization capacity and / or increased complement-mediated cell killing and / or antibody-dependent cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992). In some embodiments, the antibodies of the present invention may be antibodies having modified 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 expressing such receptors (e.g., macrophages, hepatic sinusoidal cells, etc.).

[0068] Another type of amino acid modification of the antibody of the present invention can be used to alter the original glycosylation pattern of the antibody, namely by deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites not present in the antibody. The presence of either asparagine-X-serine or asparagine-X-threonine (where X is any amino acid other than proline) in the tripeptide sequence creates potential glycosylation sites. By altering the amino acid sequence to contain one or more of the aforementioned tripeptide sequences (for N-glycosylation sites), the addition or deletion of glycosylation sites in the antibody can be conveniently achieved.

[0069] Another type of modification involves the removal of sequences identified on a computer chip (in silico) or experimentally as potentially leading to heterogeneity in degradation products or antibody formulations. As an example, deamidation of asparagine and glutamine residues can occur, depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites, especially Asn-Gly, are present in antibodies or peptides, removal of these sites can be considered, typically by conservative substitution of one of the involved residues. Such substitutions involving the removal of one or more of the involved residues from the sequence are also contemplated for coverage by this invention.

[0070] Another class of covalent modifications involves the chemical or enzymatic conjugation of glycosides to antibodies. These procedures are advantageous because they do not require antibody production in host cells that possess the glycosylation capacity for N- or O-linked glycosylation. Depending on the conjugation mode used, the sugar can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group, such as those of cysteine, (d) a free hydroxyl group, such as those of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as those of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine. Such methods are described, for example, in WO87 / 05330.

[0071] The removal of carbohydrate moieties from antibodies can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or its equivalent. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylglucosamine), while maintaining antibody integrity. Chemical deglycosylation has been described by Sojahr H. et al. (1987) and Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties from antibodies can be achieved using various endoglycosidases and exoglycosidases, as described by Thotakura, NR. et al. (1987).

[0072] Another type of covalent modification of antibodies involves linking the antibody to one of various non-protein polymers (such as polyethylene glycol, polypropylene glycol, or polyoxyethylene), for example, as shown in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 and 4,179,337.

[0073] Other amino acid sequence modifications known in the art can also be applied to the antibodies of this invention.

[0074] Anti-GD2 ADC This invention provides immunoconjugates, also referred to herein as ADCs, or more simply as conjugates. As used herein, all these terms have the same meaning and are interchangeable. The immunoconjugates of this invention can be prepared according to the in vitro methods described herein.

[0075] The present invention provides an ADC comprising an antibody of the present invention (e.g., mAb1, or an antibody having a CDR similar to mAb1) covalently linked to at least one growth inhibitor via a linker.

[0076] The term “growth inhibitor” (also known as “antiproliferator”) refers to a molecule or compound or composition that inhibits the growth of cells, such as tumor cells, in vitro and / or in vivo.

[0077] In some embodiments, the growth inhibitor is a cytotoxic drug (also known as a cytotoxic agent). The invention also considers the use of a radioactive component as a cytotoxic drug.

[0078] As used herein, the term "cytotoxic drug" refers to a substance that directly or indirectly inhibits or prevents cellular function and / or causes cellular damage. The term "cytotoxic drug" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxins, vinca, taxanes, maytansine compounds or maytansine analogs, tomatine or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, lepromycin derivatives, auristatin or dolalastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-microtubule agents, CC-1065 and CC-1065 analogs.

[0079] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in altering the topology of DNA by catalyzing transient breaks and rejoining of single strands. Topoisomerase I inhibitors are highly toxic to dividing cells, such as those in mammals. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogues such as topotecan, irinotecan, silatecan, cositecan, eczetidine, letopotecan, gemmatostatin, belotetidine, and rubitecan.

[0080] In some embodiments, the immunoconjugates of the present invention comprise the cytotoxic drug eczemab as a growth inhibitor. Ecizemab has the following IUPAC chemical name: (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]indolazino[1,2-b]quinoline-10,13-dione

[0081] Iciticon is represented by the following structural formula (I): (I) Ecinotecan is a modified derivative of camptothecin, having additional alicyclic rings fused to rings A and B, which carry a solubilizing primary amine (equivalent to the 7-CH2NH2 substituent on camptothecin). Lipophilic substituents are also present at positions 10 and 11 on ring A, enhancing membrane permeability.

[0082] In further embodiments of the invention, other CPT analogs and other cytotoxic agents, such as those listed above, may be used. Examples of some cytotoxic agents and conjugation methods are further given in application WO2008 / 010101, which is incorporated herein by reference.

[0083] The term "radioactive part" refers to a chemical entity (e.g., a molecule, compound, or composition) that contains or is composed of a radioactive isotope suitable for treating cancer, such as At. 211 Bi 212 Er 169 I 131 I 125 Y 90 In 111 P 32 Re 186 Re 188 、Sm 153 、Sr 89 Or a radioactive isotope of lutetium. Such radioactive isotopes generally emit primarily beta rays. In some embodiments, the radioactive isotope is an alpha emitter isotope, such as thorium-227, which emits alpha rays. The immunoconjugate can be prepared, for example, as described in application WO2004 / 091668.

[0084] In the immunoconjugates of the present invention, the antibody of the present invention is covalently linked to at least one growth inhibitor via a linker. As used herein, "linker" means a chemical portion comprising a covalent bond and / or covalently attaching a growth inhibitor to any atomic chain of the antibody. Linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. The conjugation of the antibody of the present invention to a cytotoxic drug or other growth inhibitor can be performed, for example, using a variety of bifunctional protein conjugates, including but not limited to N-succinimidylpyridinyl dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl butyrate (nitro-SPDB), 4-(pyridin-2-yldithio)-2-sulfonylbutyrate (sulfon-SPDB), N-succinimidyl(2-pyridinyldithio)propionate (SPDP), succinimidyl( N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminothiones (IT), bifunctional derivatives of imine esters (e.g., dimethyl adipamide HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)-hexanediamine), diazido derivatives (e.g., bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al. (1987). Carbon-labeled benzylmethyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies (WO 94 / 11026).

[0085] In embodiments of the invention, the connector may be a "cutterable connector" that facilitates the release of cytotoxic drugs or other growth inhibitors within or near cells, such as tumor cells. In some embodiments, the connector is a cuttable connector within the endosome of a mammalian cell. For example, acid-labile connectors, peptidase-sensitive connectors, esterase-labile connectors, light-labile connectors, or disulfide-containing connectors may be used (see, for example, U.S. Patent No. 5,208,020).

[0086] When referring to structural formulas representing immune conjugates, this article also uses the following nomenclature: the combined growth inhibitor and adapter are also referred to as the [(adapter)–(growth inhibitor)] part; for example, the combined eczetidine molecule and adapter are also referred to as the [(adapter)–(eczetidine)] part.

[0087] In some specific embodiments of the invention, the linker is a linker that can be cleaved by the human enzyme glucuronidase. For example, the immunoconjugate of the invention can therefore have the following formula (II), which includes a linker that can be cleaved by glucuronidase: (II), The antibody described herein is the antibody of the present invention, wherein S is a sulfur atom of the antibody, and wherein n is the number of [(connector)–(growth inhibitor)] portions covalently linked to the antibody. The number n can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​residue of the antibody. In some embodiments, the antibody is mAb1.

[0088] The number n is also known as the "drug-antibody ratio" (or "DAR"); this number n should always be understood as the average number for any given immune conjugate (of formulation).

[0089] In other specific embodiments of the invention, the linker is a linker that can be cleaved by the human enzyme asparagine endopeptidase (legumain). For example, the immunoconjugate of the invention can therefore have the following formula (III), which includes a linker that can be cleaved by asparagine endopeptidase: (III), The antibody described herein is the antibody of the present invention, wherein S is a sulfur atom of the antibody, and wherein n is the number of [(connector)–(growth inhibitor)] portions covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​residue of the antibody. In some embodiments, the antibody is mAb1.

[0090] In each of formulas (II) and (III) above, the chemical structure between the sulfur atom of the antibody and the growth inhibitor is a linker. One of these linkers is also included in each of formulas (IV) to (IX) further described below.

[0091] In any of the embodiments described above, having a linker that can be cleaved by glucuronidase or asparagine endopeptidase, the growth inhibitor may be, for example, eczema.

[0092] Accordingly, in some embodiments, the present invention provides an immunoconjugate comprising the antibody of the present invention covalently linked to eczema via a linker, wherein said conjugate has the following formula (IV): (IV), Where S is the sulfur atom of the antibody, and n is the number of [(connector)–(ecientac)] portions covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0093] In other embodiments, the present invention provides an immunoconjugate comprising an antibody of the present invention covalently linked to eczema via a linker, wherein said conjugate has the following formula (V): (V), Where S is the sulfur atom of the antibody, and n is the number of [(connector)–(ecientac)] portions covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, the antibody is mAb1.

[0094] In some embodiments, in the immunoconjugates of the present invention, such as the eczetane conjugates having a glucuronidase or asparagine endopeptidase cleavable linker as described above, the linker is covalently attached to the antibody at the sulfur atom of a cysteine ​​residue. For example, this cysteine ​​residue of the antibody can be one of the cysteine ​​residues capable of forming interchain disulfide bonds (also referred to herein as interchain disulfide bridges). Since there are four interchain disulfide bonds in IgG1 antibodies, involving a total of eight cysteine ​​residues, the attachment of the linker to the antibody at the sulfur atom of such cysteine ​​residues provides a DAR that can be as high as 8, and in such cases, the DAR is typically 7 to 8, for example 7.5 to 8.0 (i.e., about 8), provided that the antibody is IgG1 or has the same number of interchain disulfide bonds as IgG1.

[0095] Accordingly, in some embodiments, the present invention provides an immunoconjugate comprising the antibody of the present invention covalently linked to eczema via a linker, wherein said conjugate has the following formula (VI): (VI), Where S is the sulfur atom of the cysteine ​​in the antibody, and n is the number of [(linker)–(ecithecan)] portions covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8).

[0096] In other embodiments, the present invention provides an immunoconjugate comprising an antibody of the present invention covalently linked to eczema via a linker, wherein said conjugate has the following formula (VII): (VII), Where S is the sulfur atom of the cysteine ​​in the antibody, and n is the number of [(linker)–(ecithecan)] portions covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8).

[0097] In any of the above-described immunoconjugates, any antibody of the present invention (as described above and below) may be used. In some embodiments, the immunoconjugates of the present invention comprise mAb1 as an antibody.

[0098] Accordingly, in some embodiments, the present invention provides an immunoconjugate comprising mAb1 covalently linked to eczema via a linker, wherein the conjugate has the following formula (VIII): (VIII), Where S is the sulfur atom of a cysteine ​​residue in antibody mAb1, and n is the number of [(linker)–(ecithecan)] portions covalently linked to mAb1. The number n (also referred to as DAR) can be, for example, 1 to 10; in a more specific embodiment, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is the sulfur atom of a cysteine ​​residue in 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.

[0099] In other embodiments, the present invention provides an immunoconjugate comprising mAb1 covalently linked to eczema via a linker, wherein the conjugate has the following formula (IX): (IX), Where S is the sulfur atom of a cysteine ​​residue in antibody mAb1, and n is the number of [(linker)–(ecithecan)] portions covalently linked to mAb1. The number n (also referred to as DAR) can be, for example, 1 to 10; in more specific embodiments, n is 7 to 8; in even more specific embodiments, n is 7.5 to 8.0 (i.e., about 8). In some embodiments, S is the sulfur atom of a cysteine ​​residue in mAb1 capable of forming an interchain disulfide bridge, and the DAR is about 8. The number of cytotoxic agents and / or cell growth inhibitors linked to the antigen-binding portion of the anti-huLRRC15 ADC can vary (referred to as the “drug-to-antibody ratio”, or “DAR”) and is limited only by the number of attachment sites available on the antigen-binding portion and the number of agents linked to a single linker. Anti-GD2 ADCs with a DAR of 20 or even higher are considered, provided that the anti-GD2 ADC does not exhibit unacceptable levels of aggregation under use and / or storage conditions.

[0100] In other embodiments of the invention, the adapter may be an "uncuttable adapter" (e.g., an SMCC adapter). Following lysosomal degradation of the antibody, the release of growth inhibitors from the antibody may occur.

[0101] In other embodiments of the invention, the immunoconjugate may be a fusion protein comprising the antibody of the invention and a cytotoxic peptide or a growth-inhibiting peptide (as a growth inhibitor); such fusion proteins may be prepared by recombinant techniques or peptide synthesis (i.e., methods well known in the art). The DNA-encoding molecule may contain corresponding regions encoding the two parts of the conjugate (the antibody and the cytotoxic peptide or growth-inhibiting peptide, respectively), which are adjacent to each other or separated by regions encoding adaptor peptides.

[0102] In some embodiments of the invention, pain is alleviated by avoiding CDC via the Fc point mutation (K322A) in hu14.18 Ab while maintaining sufficient ADCC activity to attack tumor cells, as supported by experimental data from a rat model of anomalous pain that indicates partial relief of mechanical anomalous pain (Slart et al., 1997; Sorkin et al., 2010).

[0103] To this end, the ADC (molecule 1) was designed by conjugating a humanized ch14.18-derived Ab with eczema (a potent DNA topoisomerase I inhibitor) to induce tumor cell apoptosis following ADC internalization and the intracellular enzymatic release of the payload in lysosomes. Furthermore, both ADCC and CDC activities were modified to remove the Fc portion of the Ab to mitigate neurological damage via these mechanisms. Because, in contrast to microtubule inhibitors (Stagg et al., 2016), topoisomerase I inhibitors, either on their own (Verschraegen et al., 2000, Rowinsky 2005) or as a component of the ADC (Ogitani et al., 2016), do not induce peripheral neuropathy, cell killing of tumors with relatively high GD2 expression via the payload was achieved while sparing the peripheral nervous system (PNS) with low GD2 expression, thereby alleviating the pain effect.

[0104] The antibodies of the present invention can also be used in targeted enzyme prodrug therapies, such as antibody-targeted enzyme prodrug therapy, by conjugating the antibody with a prodrug-activating enzyme that converts the prodrug (e.g., a peptide-based chemotherapeutic agent, see WO81 / 01145) into an active cytotoxic drug (see, for example, WO 88 / 07378 and U.S. Patent No. 4,975,278). The enzyme component of the immunoconjugates usable in ADEPT may include any enzyme capable of acting on the prodrug in such a manner to convert it into its more active cytotoxic form. Enzymes useful in this context include, but are not limited to: alkaline phosphatases that can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatases that can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminases that can be used to convert non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; proteases that can be used to convert peptide-containing prodrugs into free drugs, such as Serratia marcescens protease, thermophilic protease, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L); D-alanylcarboxypeptidases that can be used to convert prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidase that can be used to convert glycosylated prodrugs into free drugs; P-lactamases that can be used to convert drugs derived from P-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derived from phenoxyacetyl or phenylacetyl groups at their amine nitrogen sites into free drugs, respectively. Enzymes can be covalently bound to the antibodies of the present invention using techniques well known in the art, such as the adapters discussed above.

[0105] Suitable methods for preparing the immunoconjugates of the present invention are well known in the art (see, for example, Hermanson GT, Bioconjugate Techniques, 3rd edition, 2013, Academic Press). For example, methods for conjugating cytotoxic drugs to antibodies via linkers covalently attached to cysteine ​​residues of interchain disulfide bridges of the antibody are well known.

[0106] Generally, the immunoconjugates of the present invention can be obtained, for example, by a method comprising the following steps: (i) Prepare compounds containing a linker and a growth inhibitor (e.g., a cytotoxic drug), also referred to herein as “drug-linker compounds”; (ii) Contacting an aqueous solution of the optionally buffered antibody according to the invention with a solution of the drug-connector compound; (iii) The conjugate formed in (ii) is then optionally separated from the unreacted antibody and / or drug-adaptor compound.

[0107] The aqueous solution of the antibody can be buffered with a buffer, such as histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer can be selected depending on the properties of the antibody. The drug-connector compound can be dissolved in, for example, a polar organic solvent such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0108] In order to conjugate with the cysteine ​​residues of the antibody, the antibody is subjected to reduction (e.g., using TCEP) prior to step (ii). Suitable reduction conditions for reducing only interchain disulfide bonds are known in the art.

[0109] The reaction temperature used for conjugation is typically 20 to 40 °C. Reaction times can vary and are generally from 1 to 24 hours. The reaction between the antibody and the drug-connector compound can be monitored by size exclusion chromatography (SEC) with a refractive index detector and / or a UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0110] A variety of different chromatographic methods can be used by those skilled in the art to perform the separation in step (iii): the conjugate can be purified, for example, by SEC, adsorption chromatography (e.g., ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed-carrier chromatography such as hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC) such as reversed-phase HPLC. Purification by dialysis, filtration, or percolation can also be used.

[0111] Following steps (ii) and / or (iii), the solution containing the conjugate may be subjected to additional purification steps (iv), such as by chromatography, ultrafiltration, and / or percolation. Where reduction is performed prior to conjugation, such additional purification steps, such as by chromatography, ultrafiltration, and / or percolation, may also be performed after the reduction reaction with the antibody-containing solution.

[0112] At the end of such a process, the conjugate is recovered in an aqueous solution. The drug-antibody ratio (DAR) is a number that can vary depending on the properties of the antibody and the drug-adaptor compound used, along with the experimental conditions used for conjugation (e.g., the ratio of (drug-adaptor compound) to (antibody), reaction time, and the nature of the solvent and co-solvent, if any). Therefore, contact between the antibody and the drug-adaptor compound can result in a mixture containing several conjugates that differ from one another due to different drug-antibody ratios. The determined DAR is therefore an average value.

[0113] Using an antibody with four inter-chain disulfide bridges (such as mAb1 or any IgG1 antibody) to perform conjugation at the cysteine ​​residues of the inter-chain disulfide bridges—a method well known in the art—offers the advantage that approximately 8 relatively homogeneous DARs can be achieved by selecting reaction conditions that allow the conjugation to proceed to completion (or at least close to completion).

[0114] An exemplary method that can be used to determine DAR consists of the following: measuring the concentration of a purified conjugate solution in λ using spectrophotometry. D The ratio of absorbance at 280 nm to absorbance at 280 nm. 280 nm is a wavelength commonly used to measure protein concentration, such as antibody concentration. Choosing wavelength λ D In order to allow for the differentiation between drugs and antibodies, i.e., as is readily known to a technician, λ D It is the wavelength at which the drug has high absorbance, and λ D The absorbance peaks of the drug and antibody are sufficiently far from 280 nm to avoid significant overlap. For example, λ D For eczema (or camptothecin or other camptothecin analogues), 370 nm can be selected, or for maytansine-like compounds, 252 nm can be selected.

[0115] Methods for calculating DAR can be derived, for example, from Antony S. Dimitrov (ed.), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, 445, Springer Science: Measuring conjugates at λ using size exclusion chromatography (SEC) analysis of monomer peaks (allowing the calculation of the “DAR(SEC)” parameter) or using classical spectrophotometer instruments (allowing the calculation of the “DAR(UV)” parameter). D(A λD ) and 280 nm (A 280 The absorbance at () is the value of light emitted at a given point. Absorbance can be expressed as follows: A λD = (C D X ε DλD ) + (C A X ε AλD ) A 280 = (C D X ε D280 ) + (C A X ε A280 ) in: C D and C A Concentrations in drug and antibody solutions, respectively. ε DλD and ε D280 The drugs in λ D and the molar extinction coefficient at 280 nm ε AλD and ε A280 Antibodies at λ D and the molar extinction coefficient at 280 nm.

[0116] Solving these two equations, each with two unknowns, yields the following equation: C D = [( ε A280 XA λD ) - (ε AλD XA 280 )] / [(ε DλD X ε A280 ) - ( ε AλD X ε D280 )] C A = [A 280 - (C D X ε D280 )] / ε A280 Then, the average DAR is calculated based on the ratio of drug concentration to antibody concentration: DAR = C D / C A .

[0117] Exemplary methods for preparing the immunoconjugates of the present invention are described in the embodiments.

[0118] Drug-connector compounds This invention also provides compounds comprising a linker and a growth inhibitor (e.g., a cytotoxic drug), also referred to herein as "drug-linker compounds". For example, this invention provides compounds having the following formula (X): (X) Or its physiologically acceptable salt; the compound is also referred to herein as “drug-connector compound 1”, “compound DL1” or “DL1”.

[0119] The present invention also provides compounds having the following formula (XI): (XI) Or its physiologically acceptable salt; the compound is also referred to herein as “drug-connector compound 2”, “compound DL2”, or “DL2”.

[0120] These drug-connector compounds can be used to prepare the immunoconjugates of the present invention as described above and below.

[0121] The drug-connector compounds of the present invention (e.g., those of formula (X) or (XI) described above) can be prepared by chemical synthesis, as further described in the examples below.

[0122] Pharmaceutical Composition The antibodies or immunoconjugates of the present invention can be combined with pharmaceutically acceptable carriers, diluents and / or excipients, and optionally with sustained-release matrices to form pharmaceutical compositions, said sustained-release matrices including, but not limited to, various classes of biodegradable polymers, non-biodegradable polymers, lipids or sugars.

[0123] Therefore, another aspect of the present invention relates to pharmaceutical compositions comprising the antibody or immunoconjugate of the present invention and pharmaceutically acceptable carriers, diluents and / or excipients.

[0124] "Drug" or "pharmaceutically acceptable" means a molecular entity and composition that, when administered to mammals, especially humans (as the case may be), does not produce adverse, allergic, or other undesirable reactions. Pharmaceutically acceptable carriers, diluents, or excipients refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0125] As used herein, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial agents, and antifungal agents. Examples of suitable carriers, diluents, and / or excipients include, but are not limited to, one or more of the following: water, amino acids, saline, phosphate-buffered saline, buffered phosphate, acetate, citrate, succinate; amino acids and their derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts such as NaCl or calcium chloride; sugars or polyols such as dextran, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, and combinations thereof. In many cases, it is useful to include isotonic agents such as sugars, polyols, or sodium chloride in the pharmaceutical composition, and the formulation may also contain antioxidants such as tryptamine and / or stabilizers such as Tween 20.

[0126] The form, route of administration, dosage, and regimen of a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, and the patient's age, weight, and sex.

[0127] The pharmaceutical compositions of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration.

[0128] In one embodiment, the pharmaceutical composition contains a medium that is pharmaceutically acceptable for an injectable formulation. These may be isotonic, sterile saline solutions (sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dried compositions, especially lyophilized compositions, which, depending on the circumstances, allow the composition to form an injectable solution after the addition of sterile water or physiological saline.

[0129] The pharmaceutical composition can be administered via a pharmaceutical assembly device.

[0130] The dosage used for administration can be adjusted based on various parameters, and for example, according to the administration method used, the relevant pathological condition, or alternatively, the desired duration of treatment.

[0131] In order to prepare a pharmaceutical composition, an effective amount of the antibody or immunoconjugate of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0132] Suitable drug forms for injection include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions; in all such cases, the form must be sterile and injectable without degradation by a suitable device or system for delivery, and it must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

[0133] The active compound, as a solution of a free base or a pharmacologically acceptable salt, can be prepared in water by appropriate mixing with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth.

[0134] The antibodies or immunoconjugates of the present invention can be formulated into pharmaceutical compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of proteins), which are formed from inorganic acids such as hydrochloric acid or phosphoric acid, or such organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, as well as such organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, etc.

[0135] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Suitable flowability can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the dispersed state, and using surfactants. Prevention of microbial activity can be achieved using various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In some cases, it may be desirable to include isotonic agents, such as sugars or sodium chloride. Extended absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0136] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound into a suitable solvent having any other components listed above (as needed), followed by filtration sterilization. Generally, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile medium containing a basic dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any other desired components from its previously sterile filtered solution.

[0137] The preparation of more concentrated or highly concentrated solutions for direct injection was also considered, with the envisioned use of DMSO as a solvent to result in extremely rapid penetration, delivering high concentrations of the active agent to small tumor areas.

[0138] After preparation, the solution can be administered in a manner compatible with the dosage form and in an amount that is therapeutically effective. The formulation is readily administered in various dosage forms (e.g., injectable solutions of the type described above), but may also be administered in the form of drug-release capsules, etc.

[0139] For parenteral administration in aqueous solutions, the solution may be appropriately buffered, and the solution should first be made isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used according to this disclosure will be known to those skilled in the art. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion, or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences," 15th edition, pp. 1035-1038 and 1570-1580). Some variations in dosage will inevitably occur depending on the condition of the subject being treated. In any case, the individual responsible for administration will determine the appropriate dose for the individual subject.

[0140] The antibody or immunoconjugate of the present invention can be formulated in a therapeutic mixture to contain, for example, about 0.01 to 100 mg per dose.

[0141] Other pharmaceutically acceptable forms, besides those formulated for parenteral administration, such as intravenous or intramuscular injection of antibodies or immunoconjugates, include tablets or other solids for oral administration, timed-release capsules, and any other currently used forms.

[0142] In some embodiments, liposomes and / or nanoparticles are considered for the introduction of peptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0143] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (around 0.1 μm in size) are typically designed using polymers that are biodegradable in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles, or biodegradable polylactic acid or polylactic acid-glycolic acid copolymer nanoparticles that meet these requirements are considered for use in this invention, and such particles can be readily prepared by those skilled in the art.

[0144] Liposomes can spontaneously form multilayered concentric bilayered vesicles (also known as multilayered vesicles (MLVs)) from phospholipids dispersed in an aqueous medium. MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayered vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution in the core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0145] In addition to the examples mentioned above, further drug forms such as nanoparticles, microparticles and capsules, implants (e.g., lipid implants) or self-curing or self-emulsifying systems have been considered.

[0146] Treatment methods and uses The inventors have discovered that the antibodies of the present invention (e.g., mAb1) can be partially internalized as GD2-antibody complexes upon binding. Furthermore, target-mediated uptake of such antibodies conjugated to a cytotoxic drug (in a preferred embodiment, eczema) has been shown to result in increased in vitro cytotoxic potency in tumor cells that have transformed into GD2-expressing cells, compared to control ADCs or target-negative cells respectively. The inventors have also demonstrated that these immunoconjugates of the present invention induce significant antitumor activity in vivo when administered with a single or multiple injection at doses ranging from 0.25 mg / kg to 10 mg / kg. Indeed, the immunoconjugates of the present invention have shown broad activity in a large collection of in vitro and in vivo models derived from various tumor types. Furthermore, the immunoconjugates of the present invention were well tolerated in dose-range exploration studies in non-human primates. These preclinical data indicate a favorable therapeutic window for subsequent clinical testing. Therefore, the antibodies, immunoconjugates, and pharmaceutical compositions of the present invention can be used to treat cancers expressing GD2.

[0147] Accordingly, the present invention provides antibodies, immunoconjugates, or pharmaceutical compositions of the present invention for use as pharmaceutical agents. For example, the present invention provides antibodies, immunoconjugates, or pharmaceutical compositions of the present invention for treating cancer. The present invention further provides a method of treating cancer, comprising administering the antibody, immunoconjugate, or pharmaceutical composition of the present invention to a subject in need of such treatment.

[0148] The cancer to be treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention is preferably a cancer expressing GD2, more preferably a cancer overexpressing GD2 compared with normal (i.e., non-tumor) cells of the same tissue origin. Cellular expression of GD2 can be readily determined, for example, by using the antibodies according to the present invention (or commercially available anti-GD2 antibodies), as described in the following section "Diagnostic Uses," and, for example, by immunohistochemical methods.

[0149] Depending on the tumor type, GD2 is involved in tumor development and malignant phenotypes through enhanced cell proliferation, motility, migration, adhesion, and invasion. This provides a principle for targeting disialotetrahexosylganglioside GD2 in cancer therapies for any tumor type overexpressing GD2. While the invention is not intended to be limited to any particular mechanism of action, it is known that anti-GD2 monoclonal antibodies target tumor cells expressing GD2, leading to phagocytosis and destruction via antibody-dependent cell-mediated cytotoxicity, lysis via complement-dependent cytotoxicity, and apoptosis and necrosis directly induced by cell death. Additionally, anti-GD2 monoclonal antibodies can also prevent circulating malignant cells from homing and adhering to the extracellular matrix.

[0150] In some embodiments, the cancers to be treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention are neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, and soft tissue sarcoma. In selected embodiments, the cancers to be treated with the antibodies, immunoconjugates, or pharmaceutical compositions of the present invention are neuroblastoma and osteosarcoma.

[0151] The antibodies or immunoconjugates of the present invention can be used alone or in combination with any suitable growth inhibitor in cancer therapy.

[0152] As described above, the antibodies of the present invention can be conjugated (linked) to growth inhibitors. Therefore, the antibodies of the present invention can be used to target the growth inhibitors to cancer cells that express or overexpress GD2 on their surface.

[0153] It is also well known that therapeutic monoclonal antibodies can lead to the depletion of cells carrying antigens specifically recognized by the antibody. This depletion can be mediated by at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent cleavage, and direct inhibition of tumor growth through signaling mediated by antibody-targeted antigens.

[0154] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which an antibody binding to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells. To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, such as the one described in U.S. Patent Nos. 5,500,362 or 5,821,337.

[0155] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system to an antibody that binds to its homologous antigen. To evaluate complement activation, a CDC assay can be performed, for example as described by Gazzano-Santoro et al. (Journal of Immunological Methods. March 1997; 202(2):163-171).

[0156] In some embodiments, the antibody of the present invention may be an antibody having a modified amino acid sequence that results in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors (e.g., macrophages, hepatic sinusoidal cells, etc.).

[0157] One aspect of the present invention relates to a method of treating cancer, comprising administering a therapeutically effective amount of the antibody, immunoconjugate, or pharmaceutical composition of the present invention to a subject in need of such treatment.

[0158] In the context of this invention, as used herein, the term "treating" or "treatment" means reversing, alleviating, inhibiting, or preventing the progression of a disease or condition, or one or more symptoms of such a disease or condition. As used herein, the term "treating cancer" means inhibiting the growth of malignant cells in a tumor and / or the progression of metastases from said tumor. Such treatment can also lead to the regression of tumor growth, i.e., a measurable reduction in tumor size. For example, such treatment can lead to the complete regression of a tumor or metastasis.

[0159] In the context of the therapeutic applications of this invention, the terms "subject" or "patient" or "subject with need" or "patient with need" refer to a subject (e.g., a human or non-human mammal) affected by or likely to be affected by a tumor. For example, the patient may be a patient identified, for instance, as sensitive to a therapeutic agent targeting GD2, particularly an antibody or immune conjugate according to the invention, as described herein.

[0160] "Therapeutic effective dose" means an amount sufficient to treat the cancerous disease in a way that provides a reasonable benefit / risk ratio suitable for any medical treatment. However, it should be understood that the total daily dosage of the antibodies, immunoconjugates, and pharmaceutical compositions of the present invention (collectively, "therapeutic agents") will be determined by the attending physician within the bounds of reasonable medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific therapeutic agent used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and the rate of excretion of the specific therapeutic agent used; the duration of treatment; drugs used in combination with or concurrently with the specific therapeutic agent used; and similar factors well known in the medical field. For example, it is well known within the art that a dose of the compound is started at a level below that required to achieve the desired therapeutic effect, and the dose is gradually increased until the desired effect is achieved.

[0161] The antibodies, immunoconjugates, or pharmaceutical compositions of the present invention can also be used to inhibit the progression of cancer metastasis.

[0162] The antibodies, immune conjugates, or pharmaceutical compositions of the present invention can also be used in combination with any other therapeutic intervention for the treatment of cancer (e.g., adjuvant therapy) and / or to reduce the growth of metastatic cancer. For example, other therapeutic interventions used in such combinations could be standard of care (SOC) treatments for the cancer to be treated.

[0163] The efficacy of treatment with the antibody or immunoconjugate or pharmaceutical composition according to the invention can be determined in vivo, for example, in a mouse model of cancer, and can be readily measured by measuring, for example, changes in tumor volume, % tumor regression, partial regression, or complete regression between the treatment group and the control group.

[0164] Diagnostic uses GD2 has been reported to be highly expressed on the surface of cancer cells such as neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, soft tissue sarcoma, or other solid tumors that express GD2.

[0165] Therefore, GD2 can be considered a cancer marker and has the potential to be used to indicate the effectiveness of anti-cancer therapies or to detect disease recurrence.

[0166] In one embodiment, the antibody of the present invention can be used as a component of an assay in the context of a therapy targeting tumors expressing GD2, in order to determine a patient's sensitivity to the therapeutic agent, monitor the effectiveness of the anticancer therapy, or detect disease recurrence after treatment. In some embodiments, the same antibody of the present invention can be used both as a component of a therapeutic agent and as a component of a diagnostic assay.

[0167] Therefore, a further aspect of the invention relates to the use of the antibody according to the invention for the in vitro detection of GD2 expression in biological samples from a subject. Another aspect of the invention relates to the use of the antibody according to the invention for the in vivo detection of GD2 expression in a subject. When used for GD2 detection, the antibody can be labeled with a detectable molecule such as a fluorophore or an enzyme.

[0168] GD2 detection can be expected to be used for: a) Diagnosing the presence of cancer in the subject, or b) Determine the sensitivity of patients with cancer to therapeutic agents targeting GD2, particularly the antibodies or immunoconjugates according to the present invention, or c) Monitoring the effectiveness of anti-GD2 cancer therapy or detecting cancer recurrence after anti-GD2 cancer therapy, particularly the therapy using antibodies or immune conjugates according to the invention; this is done by detecting the expression of surface GD2 on tumor cells.

[0169] In some embodiments, the antibodies of the present invention are intended for in vitro or ex vivo diagnostic uses. For example, GD2 can be detected in vitro or ex vivo using the antibodies of the present invention in biological samples obtained from a subject. Use according to the invention can also be in vivo. For example, the antibodies of the present invention can be administered to a subject, and antibody-cell complexes can be detected and / or quantified, whereby the detection of said complexes indicates cancer.

[0170] The present invention further relates to an in vitro or ex vivo method for detecting the presence of cancer in a subject, comprising the following steps: (a) In particular, the biological sample is contacted with the antibody according to the invention under conditions suitable for forming a complex with the antibody and the biological sample from the subject; (b) Measure the level of antibodies binding to the biological sample; and (c) The presence of cancer is detected by comparing the measured levels of binding antibodies to a control; an increase in the level of binding antibodies compared to the control indicates cancer.

[0171] The present invention also relates to an in vitro or ex vivo method for determining the sensitivity of patients with cancer to therapeutic agents targeting GD2, particularly antibodies or immune conjugates according to the present invention, said method comprising the following steps: (a) In particular, the biological sample is contacted with the antibody according to the invention under conditions suitable for forming a complex with the antibody and a biological sample from a patient with cancer; (b) Measure the level of antibodies binding to the biological sample; and (c) The measured level of the antibody bound to the biological sample is compared with the level of the antibody bound to the control; wherein an increase in the level of the antibody bound to the biological sample compared to the control indicates that the patient is sensitive to a therapeutic agent targeting GD2.

[0172] In the method described above, the control can be a normal, non-cancerous biological sample of the same type, or a representative reference value for antibody binding levels in a normal biological sample of the same type.

[0173] In one embodiment, the antibody of the present invention can be used to diagnose cancers expressing GD2, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors expressing GD2.

[0174] The present invention further relates to an in vitro or ex vivo method for monitoring the effectiveness of anti-GD2 cancer therapy, comprising the following steps: (a) In particular, the biological sample is contacted with the antibody according to the invention under conditions suitable for forming a complex with the antibody and a biological sample from a subject who has undergone anti-GD2 cancer treatment; (b) Measure the level of antibodies binding to the biological sample; and (c) Compare the measured levels of bound antibodies with the levels of bound antibodies in the control group; A decrease in the level of antibodies binding to the biological sample compared to a control indicates the effectiveness of the anti-GD2 cancer treatment. In the same method, an increase in the level of antibodies binding to the biological sample compared to a control indicates the ineffectiveness of the anti-GD2 cancer treatment. In one embodiment of this method for monitoring effectiveness, the control is a biological sample of the same type as the biological sample submitted for analysis, but obtained from the subject at an earlier time point during the anti-GD2 cancer treatment process.

[0175] The present invention further relates to an in vitro or ex vivo method for detecting cancer recurrence after anti-GD2 cancer treatment, comprising the following steps: (a) In particular, under conditions suitable for the formation of a complex between the antibody and a biological sample from the subject, the biological sample is contacted with the antibody according to the invention, the subject having completed anti-GD2 cancer treatment; (b) Measure the level of antibodies binding to the biological sample; and (c) The measured level of binding antibody is compared with the level of binding antibody in a control; wherein an increase in the level of antibody binding the biological sample compared to the control indicates cancer recurrence following anti-GD2 cancer treatment. The control may be a biological sample of the same type as the biological sample submitted for analysis, but the sample was previously obtained from the subject, i.e., at or after the completion of anti-GD2 cancer treatment.

[0176] The anti-GD2 cancer treatment is a treatment using antibodies or immunoconjugates according to the present invention. The anti-GD2 cancer treatment targets cancers expressing GD2, such as neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma, and soft tissue sarcoma, or other solid tumors expressing GD2.

[0177] In some embodiments, the antibodies of the present invention may be labeled with detectable molecules or substances, such as fluorescent molecules or fluorophores, radioactive molecules, enzymes, or any other label known in the art that provides a signal (directly or indirectly).

[0178] As used herein, the term "labeled" in relation to antibodies according to the invention is intended to encompass the direct labeling of antibodies by conjugating (i.e., physically linking) a detectable substance such as a radioactive reagent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indigo (Cy5)) to a polypeptide, as well as the indirect labeling of polypeptides by their reactivity with the detectable substance.

[0179] The antibodies of this invention can be labeled with radioactive molecules using any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms used in scintillation scanning studies, such as I... 123 I 124 In 111 Re 186 Re 188 、Tc 99 The antibodies of the present invention can also be labeled with spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0180] "Biological samples" encompass a wide range of sample types obtained from subjects 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 samples or tissue cultures or cells derived therefrom, and their progeny. Therefore, biological samples include clinical samples, cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples such as tumor samples.

[0181] In some implementations, the biological sample may be a formalin-fixed and paraffin-embedded (FFPE) or frozen tissue sample.

[0182] The present invention also relates to an in vivo method for detecting the presence of cancer in a subject, comprising the following steps: a) Administering the antibody according to the invention to a patient, wherein the antibody is labeled with a detectable molecule; b) The localization of the antibody in the patient is detected by imaging, for example by detecting detectable molecules.

[0183] In the method, the cancer can be a cancer that expresses GD2, such as neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung cancer, breast cancer, glioma, osteosarcoma and soft tissue sarcoma, or other solid tumors that express GD2.

[0184] The antibodies of this invention can also be used for cancer staging (e.g., in radiographic imaging). They can be used alone or in combination with other cancer markers.

[0185] As used herein, the terms “detection” or “detected” include qualitative and / or quantitative detection (i.e., measurement level), with or without a reference to a control.

[0186] In the context of this invention, as used herein, the term "diagnosis" means determining the nature of a medical condition based on a large amount of collected data, with the aim of identifying pathological conditions affecting the subject.

[0187] Reagent test kit Finally, the present invention also provides kits comprising at least one antibody or immunoconjugate of the present invention. Kits containing the antibodies of the present invention can be used for the detection of surface protein GD2, or for use in therapeutic or diagnostic assays. Kits of the present invention may contain antibodies conjugated to a solid support, such as a tissue culture plate or beads (e.g., agarose beads). Kits containing antibodies for the in vitro detection and quantification of surface protein GD2, for example, in ELISA or Western blotting, can be provided. Such antibodies for detection may be labeled, for example, with fluorescent or radioactive labels.

[0188] Sequence Summary amino acid sequence: Primary structure of hu14.18-IgG1.4(K322A)-delK antibody: Heavy chain sequence (SEQ ID NO:4): Light chain sequence (SEQ ID NO:1): Preferred implementation scheme of GD2 ADC In a preferred embodiment, the present invention describes a novel ADC drug that has been fully optimized according to the following overview: Antibodies with suitable affinity, good cell binding ability, and efficient internalization were selected to ensure adequate payload deployment after binding to cancer cells. The selected antibody, hu14.18-IgG1.4(K322A)-delK, is based on the known CDR sequence of dartuximab (ch14.18), but was further optimized through humanization to reduce the risk of immunogenicity in patients.

[0189] The antibody portion contains the HC K322A mutation that eliminates complement activation. This mutation reduces pain as demonstrated in a pilot trial of a humanized anti-GD2 monoclonal antibody (hu14.18K322A) in combination with other drugs, including chemotherapy.

[0190] The antibody portion undergoes a sequence modification (IgG1.4 form), which virtually eliminates binding to most Fcγ receptors. Preclinical tolerability studies in non-human primates (detailed below) confirmed that this modification, along with the K322A mutation, is maximally responsible for the near-complete reduction in pain effects and neurological damage. Therefore, as a promising therapeutic agent, this type of IgG1.4 modification in the ADC could reduce uptake and associated toxicity in other normal cells and tissues expressing Fcγ receptors, such as macrophages and hepatic sinusoidal cells. In a preferred embodiment, the antibody portion is conjugated to eczema, a highly potent topoisomerase I inhibitor, to improve antitumor activity compared to naked anti-GD2 antibody therapy. In contrast to other ADC payloads such as tubulin toxins (e.g., aurestatin and maytansine), this preferred ADC carries a lower risk of associated neurotoxicity. This is particularly advantageous when the ADC target is located on the surface of peripheral nerves.

[0191] (5) The linker design for connecting drugs and antibodies is optimized to maximize systemic stability after parenteral administration. Furthermore, the payload-linker portion of the glucuronide-based linker system ensures good conjugability, favorable PK properties, and minimal nonspecific uptake in target-negative cells and tissues.

[0192] (6) The eczema payload of the ADC used in this invention has shown a combined effect of DNA damage and response inhibitors, such as ATR / ATM inhibitors, and immune checkpoint inhibitors, which may provide additional benefits to patients in subsequent clinical studies.

[0193] Example Example 1A: Synthesis of drug-connector compounds with glucuronide-based linkers: drug-connector compounds Object 1 (DL1) Synthetic route of compound 9 (also referred to in this paper as drug-connector compound 1 (DL1)).

[0194] Chemical preparation scheme Step 1: Compound 1 To a stirred solution of (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromo-tetrahydro-pyran-2-carboxylate (8.30 g; 20.90 mmol; 1.00 equivalent) and 4-hydroxy-3-nitrobenzaldehyde (5.24 g; 31.35 mmol; 1.50 equivalent) in acetonitrile (83.00 ml; 10.00 V), silver oxide (I) (9.69 g; 41.80 mmol; 2.00 equivalent) was added. The reaction mixture was stirred at RT for 16 hours. The reaction mixture was filtered through diatomaceous earth. The filtrate was concentrated under vacuum to give a solid. The solid was dissolved in EtOAc and washed with 10% NaHCO3 aqueous solution to remove excess 4-hydroxy-3-nitrobenzaldehyde. The organic layer was concentrated under vacuum to give compound 1 as a sand-colored solid.

[0195] Yield: 9.0 g Percentage yield: 89.1% Analyze data : NMR: 1 H-NMR (400 MHz, 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 = 10.00 Hz, 1H), 5.51-5.44 (m, 1H), 5.20-5.09 (m, 2H), 4.80 (d, J = 13.20 Hz, 1H), 3.64 (s, 3H), 2.09 (s, 9H).

[0196] Step 2: Compound 2 To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 equivalent) in 2-propanol (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 equivalent) was added, followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 equivalent). The reaction mixture was stirred at RT for 1 hour. After completion, the reaction mixture was quenched with cooled H2O and filtered through diatomaceous earth. The filtrate was extracted with dichloromethane and dried over Na2SO4. The solvent was concentrated to give compound 2 as a grayish-white powder.

[0197] Yield: 8.70 g Percentage yield: 92.4% Analyze data LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 μm; Mobile phase A: H2O containing 0.1% HCOOH: ACN (95:5); B: ACN RT (minutes): 2.05; M+H: 503.2, purity: 96.6%.

[0198] Step 3: Compound 3 Palladium on carbon (10% w / w) (2.50 g; 2.35 mmol; 0.14 equivalents) was added to a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 equivalents) in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V). The reaction mixture was stirred at RT under a hydrogen atmosphere for 3 hours. After completion, the reaction mixture was filtered through diatomaceous earth. The solvent was concentrated under vacuum to give compound 3 as a grayish-white solid.

[0199] Yield: 8.5 g Percentage yield: 100% Analyze the data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 μm; Mobile phase A: H2O containing 0.1% HCOOH: ACN (95:5); B: ACN RT (minutes): 1.73; M+H: 456.10, purity: 95.1%.

[0200] Step 4: Compound 4 At 0 °C, ethyl 2-ethoxy-2H-quinoline-1-carboxylate (15.65 g; 62.66 mmol; 3.00 equivalent) was added to a stirred solution of compound 3 (7.60 g; 25.06 mmol; 1.20 equivalent) in DCM (250.00 ml; 25.00 V). The reaction mixture was stirred at RT for 16 hours. 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 a purity of 80%. The compound was further purified by washing with 30% EtOAc and petroleum ether to give compound 4 as a white solid.

[0201] Yield: 8.5 g Percentage yield: 50.7% Analyze the data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 μm; Mobile phase A: H2O containing 0.1% HCOOH: ACN (95:5); B: ACN RT (minutes): 3.03; M+H: 735.2, Purity: 81.9%.

[0202] Step 5: Compound 5 At 0 °C, bis(4-nitrophenyl) carbonate (3.06 g; 9.97 mmol; 4.00 equivalent) and DIPEA (4.40 ml; 24.92 mmol; 10.00 equivalent) were added to a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 equivalent) in THF (40.00 ml; 20.00 V). The reaction mixture was stirred at RT for 12 h. After the reaction was complete, 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 provide compound 5 as a pale yellow solid.

[0203] Yield: 2.0 g Percentage yield: 84.6% Analyze the data: LCMS: Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm; Mobile phase: A: MilliQ water containing 0.1% TFA; B: ACN RT (minutes): 3.24; M+H: 900.20, purity: 94.9%.

[0204] Step 6: Compound 6 Compound 5 (1,369 g; 1.00 equivalent) was dissolved in N,N-dimethylformamide (15.00 ml), and eczemacon mesylate (679.7 mg; 1.00 equivalent), 4-methylmorpholine (0.422 ml; 3.00 equivalent) for synthesis, and 1-hydroxybenzotriazole (172.8 mg; 1.00 equivalent) were added. The reaction mixture was stirred overnight at room temperature. After stirring time, the reaction suspension turned into a brown solution. The reaction was monitored by LC-MS, which showed complete conversion of the starting materials. The reaction mixture was purified by RP rapid chromatography. The fractions containing the product were combined, concentrated under vacuum, and lyophilized overnight to provide compound 6 as a yellow solid.

[0205] Yield: 1.59 g Percentage yield: 87.5% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4.6 mm); Mobile phase A: H2O containing 0.05% HCOOH; Mobile phase B: ACN containing 0.04% HCOOH and 1% H2O; T: 40 ℃; Flow rate: 3.3 ml / min; MS: 100-2000, AMU positive ion mode; 1% -> 100% B: 0 -> 2.0 min; 100% B: 2.0 -> 2.5 min RT (minutes): 1.95; M+H: 1196.40, purity: 84.4%.

[0206] Step 7: Compound 7 Compound 6 (1,586 g; 1.00 equivalent) was dissolved in tetrahydrofuran (50.00 ml), and LiOH solution (0.1 M) (containing lithium hydroxide hydrate (281.77 mg; 6.00 equivalent) in water (67,100 ml)) was added dropwise at 0 °C. The pH was monitored during the addition. The pH should not exceed 10. The addition of LiOH solution was completed after 1.5 hours. The reaction was monitored by LC-MS, which showed complete conversion of the starting materials. 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 fraction containing the product was fractionated and lyophilized to give compound 7 as a dark yellow solid.

[0207] Yield: 728 mg Percentage yield: 54.8% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4.6 mm); Mobile phase A: H2O containing 0.05% HCOOH; Mobile phase B: ACN containing 0.04% HCOOH and 1% H2O; T: 40 ℃; Flow rate: 3.3 ml / min; MS: 100-2000, AMU positive ion mode; 1% -> 100% B: 0 -> 2.0 min; 100% B: 2.0 -> 2.5 min RT (minutes): 1.68; M+H: 1056.30, purity: 98.5%.

[0208] Step 8: Compound 8 Compound 7 (728,000 mg; 1,000 equivalents) was dissolved in N,N-dimethylformamide (20,000 ml). Piperidine (136,513 μl; 2,00 equivalents) was added, and the solution was stirred at RT for a total of 4 hours. The reaction was monitored by LC-MS, which showed complete conversion of the starting materials. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by RP rapid chromatography. The fractions containing the product were combined, the solvent was partially removed, and the product was lyophilized overnight to provide compound 8 as a yellow solid.

[0209] Yield: 706 mg Percentage yield: 100% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4.6 mm); Mobile phase A: H2O containing 0.05% HCOOH; Mobile phase B: ACN containing 0.04% HCOOH and 1% H2O; T: 40 ℃; Flow rate: 3.3 ml / min; MS: 100-2000, AMU positive ion mode; 1% -> 100% B: 0 -> 2.0 min; 100% B: 2.0 -> 2.5 min RT (minutes): 1.22; M+H: 834.30, purity: 97.6%.

[0210] Step 9: Compound 9 To a solution of compound 8 (854 mg; 1.00 equivalent) in dimethylformamide (30.00 mL), N-ethyldiisopropylamine (149,234 μl; 1.00 equivalent) and 2,5-dioxo-pyrrolidine-1-yl ester of 3-(2,5-dioxo-2,5-dihydro-pyrrolo-1-yl)-propionic acid (233.61 mg; 1.00 equivalent) were added. The reaction mixture was stirred at RT for 3 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting materials. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by RP rapid chromatography. The fractions containing the product were combined, concentrated, and lyophilized to give the desired product with a purity of 91%. This material was purified again by RP chromatography to give compound 9 as a yellow solid.

[0211] Yield: 580 mg Percentage yield: 60.1% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4.6 mm); Mobile phase A: H2O containing 0.05% HCOOH; Mobile phase B: ACN containing 0.04% HCOOH and 1% H2O; T: 40 ℃; Flow rate: 3.3 ml / min; MS: 100-2000, AMU positive ion mode; 1% -> 100% B: 0 -> 2.0 min; 100% B: 2.0 -> 2.5 min RT (minutes): 1.38; M+H: 985.30; Purity: 90% (the other 10% of isomers can be removed by HPLC) 1 H NMR (500 MHz, DMSO- d 6) δ 13.10 – 12.44 (m, 1H), 9.08 (s, 1H), 8.32 (t, J =5.8 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 10.9 Hz, 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= 7.6 Hz, 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 = 7.3 Hz, 3H).

[0212] Example 1B: Synthesis of drug-connector compounds with asparagine endopeptidase-cleavable linkers: drug-connector Head compound 2 (DL2) Step 1 {-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propamido]propamido]propamido]phenyl}methyl 4-nitrophenyl carbonate (400 mg; 0.52 mmol; 1.00 equivalent) [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-diazahexane [14.7.1.0] 2 , 1 4.04, 13 .06, 11 .0 2 °, 2 [4] Ticosac-1,6(11),12,14,16,18,20(24)-heptaen-5,9-dione; methanesulfonic acid (277.30 mg; 0.52 mmol; 1.00 equivalent), N-ethyldiisopropylamine (0.27 ml; 1.57 mmol; 3.00 equivalent) and 1-hydroxybenzotriazole (HOBT) (3.52 mg; 0.03 mmol; 0.05 equivalent). The reaction mixture was stirred overnight at room temperature. LC / MS indicated complete conversion.

[0213] 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)propamido]propamido]propamido]phenyl}methylN-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.0] ..." ] ] " ] ] ] " ] ] " ] ] ] " ] ] " ] ] " ] ] " ] ] " ] " ] " ] ] " ] " ] " ] " ] " " "" ] 10 """ 10-—" """"""""'" """" """"""" """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 2 , 1 4.04, 13 .06, 11 .0 2 °, 2 4] TCO-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate.

[0214] LC / MS: [M+H] = 1064.2 Preparative HPLC: Column: Sunfire Prep C18 OBD - 75.0 g (250 bar) Solvent A: Wasser 0.1% TFA Solvent C: Solvent B: Acetonitrile 0.1% TFA. Step 2 The formula {4-[(2S)-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propamido]propamido]propamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.0]} is used. 2 , 1 4.04, 13 .06, 11 .0 2 °, 2 4] TCO-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (365 mg; 0.34 mmol; 1.00 equivalent) was dissolved in N,N- (4.00 ml). Piperidine (0.07 ml; 0.69 mmol; 2.00 equivalent) for synthesis was added, and the reaction solution was stirred at rt for 1 hour.

[0215] 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-aminopropamido]propamido]-3-carbamoylpropamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.0] 2 , 1 4.04, 13 .06, 11 .0 2 °, 2 4] TCO-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate.

[0216] LC / MS: [M+H]: 841.3 Preparative HPLC for purification: RediSep Säule: C18 130g SN: E0410A0D24BE1 Batch: 262118923W Flow rate: 75 ml / min Step 3 To trifluoroacetic acid {4-[(2S)-2-[(2S)-2-[(2S)-2-aminopropamido]propamido]-3-carbamoylpropamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.0] 2 , 1 4.04, 13 .06, 11 .0 2 °, 2 4] Ticocarbamate-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamate (571 mg; 0.60 mmol; 1.00 equivalent) was added to a solution of N,N-dimethylformamide (20 mL), along with N-ethyldiisopropylamine (203 µL; 1.20 mmol; 2.00 equivalent) and N-succinimide-3-maleimide propionate (162 mg; 0.60 mmol; 1.00 equivalent). The reaction mixture was then stirred for 10 minutes and monitored by LC / MS.

[0217] The reaction mixture was purified by preparative HPLC to give 378 mg (0.35 mmol) of DL2.

[0218] LC / MS: [M+H]: 992.4 Preparative HPLC for purification: RediSep column: C18 86g SN: E0410A8B46130 Batch: 281729189W Flow rate: 60 ml / min Conditions – Volume: 264.0 ml Elution buffer 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 T: 40℃ | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive ion mode Column: Chromolith HR RP-18e 50-4,6 mm 0% -> 100% B: 0 -> 2,0 minutes | 100% B: 2,0 -> 2,5 minutes.

[0219] Example 2: Preparation of immunoconjugates: glucuronide-based mAb1 conjugates (referred to as ADC1) Antibody preparation and conjugation Monoclonal antibodies (mAbs) were thawed at 2–8°C for up to 3 days prior to conjugation and stored at 2–8°C until use. On the day of conjugation, the mAbs were equilibrated at room temperature prior to use. The pH of the mAb solution (25.3 mg / mL) at pH 5.5 was adjusted by adding 4% v / v 0.5 M Tris, 0.025 EDTA, and pH 8.5. Subsequently, the pH-adjusted mAb solution was incubated with 5 molar equivalents (relative to the mAb) of TCEP at 20°C for 2 hours to reduce interchain disulfide bonds. Afterward, the solution was incubated with 10 molar equivalents (relative to the mAb) of drug-linker (DL) at 20°C for 1 hour to allow DL conjugation to the antibody. The reaction was stopped by adding 10 molar equivalents (relative to the mAb) of 20 mM N-acetylcysteine ​​(NAC) for 20 minutes at 20°C. The pH was adjusted again by adding 5% v / v 0.15 M acetic acid, and the mixture was vacuum filtered using a 0.22 µm PES filter unit. The reaction mixture was prepared using Ultracel® Membrane, C Screen, 0.1m... 2 The Pellicon® Capsule is percolated via TFF. As the percolation buffer, 10 mM histidine and 40 mM NaCl (at pH 5.5) are used, and a flow rate of 5 L / min / m³ is required. 2 The percolation volume (DV) was 15. 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 of 10 mM histidine, 40 mM NaCl, 6% trehalose, at pH 5.5.

[0220] Quality attributes: Active pharmaceutical ingredient characterization Size exclusion chromatography (SEC) Monomer content and purity were evaluated by size exclusion chromatography on a TOSOH TSKgel G3000SWXL 7.8 mm × 30 cm, 5 μm column with a safety guard, run at 0.5 ml / min for 30 min isocratic. Injection volumes ranged from 1 to 10 μl (up to 20 μg protein).

[0221] SEC method parameters wavelength 280 nm Column TOSOH TSKgel G3000SWXL 7.8 mm × 30 cm, 5 μm Mobile phase: 10% IPA, 0.2M potassium phosphate, 0.25M potassium chloride, pH 6.95 Injection volume: 1-20 μL (5-20 µg of protein) Column temperature 25℃ Gradient isocratic elution Flow rate 0.5 mL / min Running time 30 minutes Figure 22 The document provides a typical SEC chromatogram showing the purity of the original mAb and the final BDS.

[0222] for Figure 22 The BDS material shown has been reported to have 4.6% HMWS and 95.4% monomer purity.

[0223] Reversed-phase HPLC (RP HPLC) Reversed-phase analysis 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-minute linear gradient between 0.1% TFA 25% CH3CN (mobile phase B) and 0.1% TFA 50% CH3CN (A). Both the reduced antibody and the fully conjugated ADC were analyzed under non-reduced PLRP conditions. The methods used for sample preparation are given in the table below: Figure 23 The paper provides a typical RP-HPLC chromatogram showing the separation of light and heavy chains. Figure 23 The chromatogram shows the superposition of mAb and final BDS: for Figure 23 The BDS material shown reported a DAR of 7.8.

[0224] Hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) with gradient elution and detection by absorbance at 214 nm was used to evaluate the mean DAR of the ADCs. ADC molecules were characterized in ascending order of DAR by decreasing the salt concentration in the mobile phase consisting of 1.5 M ammonium sulfate / 20 mM sodium phosphate / pH 6.95. A Tosoh TSKgel Butyl-NPR column, 4.6 × 35 mm, 2.5 µm, was used as the column. Injection volumes ranged from 1 to 10 μl for antibody / conjugate samples (5–20 μg protein).

[0225] exist Figure 24 The document provides a typical HIC chromatogram showing the separation of mAb and ADC. Figure 24 The chromatogram shows the superposition of the input mAb and the final BDS: Free drug method Reversed-phase HPLC was performed on a Phenomenex Kinetex Core Shell 2.6µm C8 column, 100 Å, 50 x 4.6 mm column, running at 2 mL / min and 60 °C, with an 8-minute linear gradient between 95% A (0.05% TFA / H2O) and 95% B (0.05% TFA / CH3CN). Toxin linker standards were run as NAC quenching. Data were collected at 214 nm, and spectra were analyzed at 252, 360, and 280 nm to monitor linker chromophores and any protein residues in the samples. Prior to HPLC analysis, the samples were deproteinized by cold methanol / salt extraction: 2 μl of 5 M NaCl was added to 50 µl of sample, followed by 150 μl of ice-cold methanol. The samples were vortexed and incubated at -20 °C for 30 min. The precipitated proteins were formed into a precipitate by centrifugation at 15000 x g and 4 °C for 30 min. 125 μl of supernatant was diluted and mixed with 125 μl of Elga 18.2 MΩ water. 100 μl of sample was injected, and all data were reported at 360 nm. The levels of residual toxin-related species were calculated relative to a toxin standard curve. The observed toxin amount relative to the total toxin content of the sample was reported as a %mol / mol value, such as... Figure 25 As shown in the image.

[0226] Overlay of RP-HPLC chromatograms (360 nm) of crude conjugate reaction (blue), DV0 (red), DV6 (green), DV10 (pink) and BDS PPB-13430 (olive green).

[0227] endotoxin Endotoxin was determined using the Endosafe PTS endotoxin system (Charles River) via dynamic colorimetric LAL assay. 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.

[0228] GD2-ADC: In vitro and in vivo experimental results Experiment 1. Resistance Dose-response efficacy study in a neuroblastoma (CHP134) xenograft model The dose-response relationship of anti-GD2 ADC molecule 1 was evaluated in CHP134 (a human neuroblastoma xenograft model expressing high levels of GD2).

[0229] Female athymic nude mice (nu / nu) with established CHP134 tumors (~150 mm3) were treated with intravenous (IV) injections of molecule 1 at doses ranging from 0.25 mg / kg to 1.0 mg / kg for 4 weeks on a weekly schedule (qw).

[0230] In this study, the treatment was well tolerated, with no toxicity observed and no increase in normal weight observed. Figure 1 The effect of treatment on CHP134 tumor growth and the size of individual tumors on the day the tumors in the mediator-controlled treatment reached the study endpoint (>1,000 mm3) were studied. Figure 2 As shown in the figure. At D0 (day 0), D7, D14, and D21, administration of molecule 1 at doses of 0.25, 0.5, and 1.0 mg / kg confirmed dose-dependent antitumor activity. The lowest dose tested, 0.25, showed efficacy of 59% tumor growth inhibition rate (TGI), which was still significant (p<0.01). Figure 2 Strong tumor regression-inducing activity was observed at 0.5 mg / kg and 1.0 mg / kg molecule 1, which was highly significant at both doses (p<0.0001, day 18). Figure 2 At the end of the study (day 85), complete responses were observed in all animals (n=16 of 16) treated with molecule 1 at doses of 0.5 mg / kg and 1.0 mg / kg. Figure 3 ).

[0231] In summary, the results from this study confirm that the potent antitumor activity of molecule 1 leads to significant efficacy and tumor regression in CHP134 tumors. The minimum effective dose (MED), defined as the lowest dose that induces a >20% reduction in tumor volume from baseline (for any time point after treatment initiation), was determined to be approximately 0.5 mg / kg of molecule 1 in the CHP134 tumor model.

[0232] Experiment 2. Efficacy study of anti-GD2 ADCs in a patient-derived xenograft (PDX) model of osteosarcoma. The efficacy of anti-GD2 ADC molecule 1 was evaluated in a CTG-2735 osteosarcoma PDX model expressing GD2.

[0233] On day 0, female athymic nude mice (nu / nu) with established CTG-2735 tumors (~250 mm3) were treated with a single IV injection of molecule 1 at a dose of 10.0 mg / kg. The group size for the vector group and the treatment group was 3.

[0234] In this study, the treatment was well tolerated, with no toxicity observed and no increase in normal weight observed. Figure 4 Treatment with 10 mg / kg molecule 1 elicited strong tumor growth inhibition, leading to long-lasting tumor regression until day 62 at the end of the experiment (see, Figure 5 and 6 ).

[0235] In summary, the results of this study confirm the potent antitumor activity of molecule 1 in the osteosarcoma PDX model CTG-2735.

[0236] Experiment 3. Efficacy study of anti-GD2 ADCs in a patient-derived xenograft (PDX) model of SCLC. The efficacy of anti-GD2 ADC molecule 1 was evaluated in a CTG-0199 SCLC PDX model expressing GD2.

[0237] On day 0, female athymic nude mice (nu / nu) with established CTG-0199 tumors (~250 mm3) were treated with a single IV injection of molecule 1 at a dose of 10.0 mg / kg. The group size for the vector group and the treatment group was 5.

[0238] In this study, the treatment was well tolerated, with no toxicity observed and no increase in normal weight observed. Figure 7 Treatment with 10 mg / kg of molecule 1 caused strong tumor growth inhibition, leading to long-lasting tumor regression until day 53 at the end of the experiment. Figure 8 , 9 ).

[0239] In summary, the results of this study confirm the potent antitumor activity of molecule 1 in the SCLC PDX model CTG-0199.

[0240] Experiment 4. Combination efficacy study of anti-GD2 ADC and ATR inhibitor in a patient-derived xenograft (PDX) model of osteosarcoma (CTG-2264). The antitumor activity of the ADC selected in this invention was tested in the CTG-2264 osteosarcoma PDX model.

[0241] On day 0, female athymic nude mice (nu / nu) with established CTG-2264 tumors (~250 mm3) were treated with: a single IV injection of molecule-1 at a dose of 3.0 mg / kg; an ATR inhibitor at a dose of 10 mg / kg orally (po), once daily (qd); and a combination of the two treatments. Saline was used as a mediator control and administered on day 0 (10 mL / kg). Each treatment group had a group size of 3. Tumor growth inhibition rate (TGI) was calculated on day 23 after treatment initiation, at which point the mean tumor volume in the control group reached 1000 mm3 (see [link to treatment]). Figure 11 , 12 ).

[0242] The treatment was well tolerated, with no noticeable toxicity or clinical signs, and no mean weight loss compared to initial weight was observed in any of the treatment groups. Figure 10 These ADCs, individually and in combination, resulted in tumor growth inhibition rates (TGI) of 65%, 67%, and -72% (regression) in the CTG-2264 osteosarcoma PDX model.

[0243] In summary, the combination mentioned above demonstrated a benefit compared to either of the two single treatments at 10 mg / kg (p<0.0001).

[0244] Experiment 5. Assay of ADC cell killing effect on human cancer cell lines Using CellTiter Glo ® Luminescent Cell Viability Assay (#G7573, Promega Corporation, Madison, WI, USA) measured the in vitro potency of ADC hu14.18-IgG1.4 (K322A)-delK-βGluc eczemacon and rituximab IgG1 βGluc eczemacon, unconjugated antibody hu14.18-IgG1.4 (K322A)-delK, and free eczemacon in the following cell lines: 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, LaJolla, CA, USA) and antigen-negative human tumor cell line MDA-MB-468 (breast cancer, #ACC738, DSMZ, Braunschweig, Germany).

[0245] Cell lines CHP-134, NCI-H446, and MDA-MB-468 were cultured as directed by the supplier and maintained at GlutaMAX levels. TM Supplement (#61870-010, Gibco) TM Purchased from Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate (#11360-070, Gibco) TM Melanoma cell line M21 was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (#S0615, Sigma Aldrich, St. Louis, MO, USA). The medium contained 10% fetal bovine serum (FBS) (#41965-039, Gibco). TM They were cultured in DMEM containing 10% FBS (Thermo Fisher Scientific).

[0246] One day prior to treatment, 625 cells / well (90 µl) (CHP-134, M21, or MDA-MB-468) or 1250 cells / well (90 µl) (NCI-H446) were cultured in sterile Falcon. TM Plates were prepared in 96-well, cell culture-treated flat-bottomed microplates (#353219, Corning, NY, USA). After overnight incubation at 37°C and 5% CO2 or 10% CO2, the compound was prepared at a starting concentration of 10x and serially diluted (1:4) using cell culture medium or RPMI 1640 medium supplemented with the above-mentioned supplements. A total of 10 μl of the compound solution was added to each well (three technical replicates). Control wells were treated with separate amounts of dimethyl sulfoxide (for free payload control wells) or cell culture medium / RPMI 1640 (for ADC and unconjugated antibody control wells and cell-free background wells). After 6 days of incubation, 100 µl of Cell Titer-Glo was added to each well. ®The reagents were prepared, and the plate was incubated with shaking at 300 rpm for 2 minutes, followed by incubation at room temperature (in the dark) for an additional 20 minutes. Subsequently, the luminescence signal was measured on 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 (where RLU for untreated control cells was defined as 100%) or %effect (calculated by subtracting 100% from %viability). Processed data were described using the equation log(inhibitor) versus slope of the response variable (four parameters), with %effect versus concentration [M] used to describe the dose-response (GraphPad Prism for Windows (version 8.2.0), GraphPad software, La Jolla, California, USA, www.graphpad.com). Data are presented as error bars indicating the standard deviation (SD) of three technical replicates. Several experiments were performed, and the determined IC50 values ​​were calculated. 50 geometric mean (geometric mean IC) 50 [nM]).

[0247] The cytotoxic activity of anti-GD2 hu14.18-IgG1.4 (K322A)-delk-βGluc eczetcan ADC (molecule 1) was determined in target-positive and target-negative human cancer cell lines. Rituximab IgG1 βGluc eczetcan ADC (molecule 2) with the same linker-loador and comparable DAR, and the unconjugated antibody hu14.18-IgG1.4 (K322A)-delK (molecule 3) were used as controls. Ecinotecan (molecule 4) was also included in the assay to confirm the general sensitivity of human cancer cell lines to the free load.

[0248] The results of in vitro activity are summarized in Table 1, and representative dose-response curves are shown in Table 1. Figure 1 - As shown in the figure. Anti-GD2hu14.18-IgG1.4 (K322A)-delk-βGluc eczemacon ADC (molecule 1) shows targeting of the GD2+ expressing cancer cell line CHP-134 ( Figure 1 Molecular 1 (M1) effectively inhibited cell viability of M21 (Fig.) and NCI-H446 (Fig.), with specific sub-nanomolar or single-digit nanomolar potency, as shown relative to the unbound control ADC (M1). For the target-negative cell line MDA-MB-468 (Fig.), which showed high sensitivity comparable to the free payload eczetidine (M1), no specific cytotoxic effect was observed for molecule 1 compared to the unbound control ADC (M1).

[0249] Treatment of the GD2-positive neuroblastoma cell line CHP-134 with the unconjugated antibody hu14.18-IgG1.4 (K322A)-delk (molecule 3) showed cytotoxic activity at the highest concentration tested (Figure). This effect is consistent with data published by Horwacik et al. (Cancer Letters, Vol. 341, No. 2, 2013, pp. 248-264), who reported a negative effect on CHP-134 cell viability after treatment with anti-GD2 mouse mAb 14G2a. Treatment with hu14.18-IgG1.4 (K322A)-delk (molecule 3) had a minor effect on cell viability in the SCLC cell line NCI-H446 and the melanoma cell line M21, while no effect was observed in the target-negative MDA-MB-468 cell line. NC: Cannot be calculated due to incomplete dose-response curve. N: Number of experiments IC 50 : Half-maximal inhibitory concentration; concentration below which 50% of the maximum inhibition was observed; the table includes IC50 values ​​based on the indicated number of experiments. 50 Geometric mean IC calculated by value 50 [nM] Span (%): % efficacy at the highest tested concentration The percentage of total non-viable cells relative to untreated control cells; the table includes the geometric mean span (%) calculated based on the span (%) values ​​of the indicated number of experiments. Maximum test concentration: 100 nM for molecules 1, 2, and 4; 1 µM for molecule 3. Table 1. Geometric mean IC50 values ​​of hu14.18-IgG1.4 (K322A)-delK-βGluc ixenograft, rituximab IgG1 βGluc ixenograft, hu14.18-IgG1.4 (K322A)-delK, and ixenograft payload in human cancer cell lines. 50 Value [nM] and geometric mean span (%) Legend for Experiment 5 Figure 13 In vitro dose-response curves for hu14.18-IgG1.4 (K322A)-delK-βGluc eciletecan (molecule 1), the control ADC rituximab IgG1 βGluc eciletecan (molecule 2), and the payload eciletecan (molecule 4) in CHP-134 cells. A representative dose-response curve is shown as the mean ± SD from three replicates.

[0250] Figure 14 In vitro dose-response curves for hu14.18-IgG1.4 (K322A)-delK-βGluc eciletecan (molecule 1), the control ADC rituximab IgG1 βGluc eciletecan (molecule 2), and the payload eciletecan (molecule 4) in M21 cells. A representative dose-response curve is shown as the mean ± SD from three replicates.

[0251] Figure 15 In vitro dose-response curves for hu14.18-IgG1.4 (K322A)-delK-βGluc eciletecan (molecule 1), the control ADC rituximab IgG1 βGluc eciletecan (molecule 2), and the payload eciletecan (molecule 4) in NCI-H446 cells. A representative dose-response curve is shown as the mean ± SD from three replicates.

[0252] Figure 16 In vitro dose-response curves for hu14.18-IgG1.4 (K322A)-delK-βGluc eciletecan (molecule 1), the control ADC rituximab IgG1 βGluc eciletecan (molecule 2), and the payload eciletecan (molecule 4) in MDA-MB-468 cells. A representative dose-response curve is shown as the mean ± SD from three replicates.

[0253] Figure 17 In vitro dose-response curves for hu14.18-IgG1.4 (K322A)-delK-βGluc (molecule 1) and hu14.18-IgG1.4 (K322A)-delk (molecule 3) in CHP-134 cells. Data are presented as mean ± SD from three replicates. The curves depicted in the figure are from two independent experiments.

[0254] Experiment 6: Safety profile of ADC1: Lead IV toxicity in cynomolgus monkeys - TK study To investigate the safety profile, ADC1 was administered to cynomolgus monkeys via 30-minute intravenous infusion at doses of 4, 8, 16, and 32 mg / kg, three times at weekly intervals (on days 1, 8, and 15), and animals were sacrificed (on day 22) for gross and histopathological examination of large groups of organs and tissues, including peripheral nerve tissues. Clinical signs, body weight, clinical hematological and biochemical parameters, and toxicokinetics (TK) were also included in this study. ADC1 induced dose-dependent effects primarily in the hemolymphatic and gastrointestinal systems, similar to eczema toxicity (De Jager 2000, Verschraegen 2000, Rowinsky 2005). Notably, no clinical signs of pain were observed during cage observation, during treatment of monkeys receiving the dose, or when blood samples were collected. No histopathological changes were observed upon examination of several peripheral nerve tissues (i.e., the peroneal, tibial, and sciatic nerves) or central brain tissues. Sufficient plasma exposure levels of conjugated antibodies were shown to correlate with extremely low plasma concentrations of unconjugated (released) eczema. The absence of pain in this study is significant because pain or atypical pain is a dose-limiting toxicity when treating cancer patients with approved GD2 (IgG1) antibodies such as datuximab (ch14.18 / SP2 / 0, Unituxin), datuximab β (ch14.18 / CHO or APN311), and nalcitubab (hu3F8). Datuximab β, when tested in cynomolgus monkeys, showed clinical signs that were interpreted as acute pain, such as those confirmed by histopathological changes observed in nerve innervation structures of the peripheral nervous system or non-neural tissues (EMA / 263814 / 2017 Datuximab β Apeiron assessment report). The labeling of datuximab β, along with the original datuximab and nalcitubab antibodies, confirms the safety information regarding acute pain (treatment). Even with GD2 antibodies that target only the CDC effector via the K322A mutation, pain can only be partially relieved (Dobrenkov and Cheung 2014; Navid et al. 2014), and the use of opioids is still required (Harman et al. 2019).

[0255] Overall, the monkey safety data and mouse tumor model data using the novel ADC showed that tumors were killed by targeting eczema-mediated apoptosis (rather than CDC and / or ADCC mechanisms) without pain associated with administration of naked anti-GD2 antibody alone.

[0256] Experiment 7: Evaluation of ADC (Molecular 1) of the peripheral nervous system in rats and cynomolgus monkeys. To investigate the preliminary safety profile of molecule 1 in both monkeys and rats expressing the same GD2 glycotope that can be used to target the conjugated hu14.18 Ab variant (FDA BLA #125516, 2014; EMA / 263814 / 2017, 2017), molecule 1 showed the expected eczema toxicity profile, but did not cause any PNS damage in monkeys and rats after repeated weekly intravenous administration. Furthermore, no indication of pain signals was observed in the animals' normal behavior in their cages, such as locomotion, standing, and exploration, or at the time of treatment.

[0257] method The general toxicology program consists of a lead repeated-dose toxicity study in Wistar rats and cynomolgus monkeys using molecule 1 administered intravenously once weekly for three consecutive weeks (days 1, 8, and 15). The study includes exposure confirmation for the ADC (conjugated payload analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and total antibody analyzed by immunoassay), and toxicokinetic assessment of the payload released in plasma (analyzed by ultra-high performance LC-MS / MS). Autopsy for macroscopic and microscopic examination is planned for day 22.

[0258] Intravenous dosage preparations: Molecule 1 (manufactured by EMD Serono, Billerica, USA, and conjugated by SterlingDeeside Ltd., Flintshire, UK) was formulated in a media solution (control group) containing 10 mM histidine, 40 mM NaCl, 6% trehalose dihydrate, and 0.05% polysorbate 20 (pH 5.5) and deep-frozen (-60°C). oC) Storage until use. Before administration, the stock concentration of molecule 1 (10 mg / mL) and diluents were validated for stability and concentration (Merck KGaA, Darmstadt, Germany). The test item or mediator was administered to rats via slow intravenous (iv) bolus infusion (tail vein) and to cynomolgus monkeys via 30-minute infusion (peripheral vein). General parameters used for toxicity assessment. Toxicity indicators consisted of the following: routine clinical observation, body weight, food intake, clinicopathology, immunophenotyping, gross pathology, organ weight, and histopathology. Autopsy included examination of the cadaver; external orifices; abdominal, thoracic, and cranial cavities; and organs. Tissues collected at autopsy were preserved in 10% neutral buffered formalin, Davidson's fixative (eye and optic nerve), or modified Davidson's fixative (testis) and processed for routine histological examination (paraffin-embedded tissue and HE staining). Selected organs were weighed prior to fixation. In addition, unscheduled autopsies and histological examinations are performed on dying animals.

[0259] B. Method Toxicity and toxicokinetics in lead rats after 3 weeks Molecular 1 was administered intravenously once weekly for 3 weeks to four groups of Crl:WI (Han) rats (5 rats / sex / group; Charles River Laboratories, Sulzfeld, Germany) at doses of 0 (mediator control), 10, 30, and 60 mg / kg. Satellite groups of 3 rats / sex / group were used for toxicokinetics assessment of the three analytes. Methods: Rats were housed in groups of 2–3 rats / cage. Appearance, behavior, and clinical signs of the rats were observed daily. Body weight was recorded before treatment initiation and daily thereafter until the end of the study. Food intake was recorded for each cage by weighing unconsumed food at weekly intervals until the end of the study. Hematological and clinical chemistry tests were performed on 5 rats / sex / group one week after the last dosing period (day 22). Hematological parameters were analyzed using an ADVIA 2120i automated analyzer, and clinical chemistry parameters were analyzed using an ADVIA 1800 automated analyzer (both from Siemens Healthcare Diagnostics, GmbH). Blood samples (0.15 mL in heparin lithium tubes) from both the satellite and master groups (sparse sampling) were collected at the following time points on days 1 and 15 and up to one week thereafter for bioanalysis and toxicokinetic (TK) assessment from all animals: 0 (before dose), and 0.5, 4, 24, 48, 96 (day 1 only), and 168 hours after dose (before the next dose). As controls, animals treated with the vector were sampled on days 1 and 15 (4 hours after the first and last administration).

[0260] ii. A 3-week toxicity and toxicokinetics study in pilot monkeys, including functional evaluation of cardiovascular (CV) parameters and respiratory rate. Crab-eating macaques bred specifically for experimental use for the first time (Macaca fascicularis) The monkeys were purchased from Envigo (Venray, Netherlands). Born and bred in Vietnam, and quarantined in Camarles (Camarney SLU, Spain), they were then transported to the testing facility in Ivrea (Italy). The monkeys were housed in air-conditioned rooms (22 ± 2°C) with 15–20 air changes / hour, 55 ± 15% relative humidity, and artificial lighting, accompanied by a 12-hour diurnal cycle from 7:00 AM to 7:00 PM. Animals were housed in groups in monkey enclosures fixed to the floor. The front and top walls of the enclosures were made of stainless steel strips, while the side and rear walls were made of colored plastic material. In this study, the experimental groups consisted of one animal of each sex per group, but additional untreated animals were present to ensure socialized rearing groups with two or three subjects. The rearing of the non-human primates complied with Italian requirements for laboratory animal welfare, including environmental enrichment programs. At the start of treatment, both males and females were approximately 3–4 years old, with a weight range of 3.1–3.8 kg. Molecular 1 was administered weekly to one male and one female cynomolgus monkey in each group via 30-minute intravenous infusion at three dose levels (4, 8, and 16 mg / kg) for three consecutive weeks. An additional male monkey was infused at a fourth, higher dose level (32 mg / kg) to determine maximum tolerability.

[0261] Mortality and clinical signs (appearance, behavior, and general signs) were recorded twice daily and at the end of infusion (up to 30 minutes after the dose), and body weight was recorded weekly throughout the study period, starting one week prior to treatment. Food and water intake (per cage) was recorded daily. Hematology (ADVIA 2120i Siemens analyzer) was investigated in all monkeys during the pre-treatment period, and on day 3 (i.e., 48 hours after the first dose), day 8 (168 hours before the second dose), day 15 (before the third dose), day 17 (i.e., 48 hours after the third dose), and day 22 (before sacrifice). Immunophenotyping (FACS Lyric Becton Dickinson flow cytometry, Becton Dickinson antibody) was performed on the same day to measure total B cells (CD3+). - CD20 + ), total T cells (CD3) + ), helper T cells (CD3) + CD4 +) and cytotoxic T cells (CD3) + CD8 + ) and natural killer cells (CD3) - CD16 + Absolute and relative counts of ) were performed. Clinical chemistry (AU480 Beckman Coulter analyzer) was performed during the pre-dose period and on days 15 and 22.

[0262] Blood samples (0.6 mL in heparin lithium tubes) were collected from each animal for bioanalysis and TK assessment at the following time points on days 1 and 15 and up to one week thereafter: 0 (before dose), and 0.5 hours after dose (=end of infusion), 2, 6, 24, 48, 72, 120, and 168 hours after dose (before the next dose).

[0263] iii. Comprehensive evaluation using electrocardiogram and arterial blood pressure As an important part of the pilot monkey DRF toxicity study, heart rate (HR), electrocardiogram (ECG), and arterial blood pressure (systolic and diastolic BP) were measured in all monkeys at baseline (before administration) and within 30 minutes of the end of the last dose infusion (day 15). Animals were trained to be recorded while seated in a chair in an awake, temporarily restrained state. Three consecutive measurements of arterial systolic and diastolic blood pressure were performed before the respiratory rate and ECG measurements. An assessment was performed on at least 10 representative ECG complexes. Respiratory rate was measured immediately after arterial blood pressure measurement by counting respiratory movements over one minute. Rectal temperature was recorded in all animals twice before administration, and on days 1, 8, and 15, at the end of the infusion (within 30 minutes after the dose) and 24 hours (±30 minutes) after the dose.

[0264] Methods: ECG electrodes (disposable foam electrodes) were placed on each animal according to standard lead II. Once the heart rate stabilized, the signals were digitized using a software package (Ponemah Physiology Platform 5.20, from DSI vendor) (A / D converter ACQ-7700, DSI, St. Paul, Minnesota, USA) and recorded continuously. Cuffs for arterial blood pressure (BP) measurement were connected to a blood pressure monitor (CARESCAPETMV100 vital signs monitoring unit, GE Healthcare, Milwaukee, Wisconsin, USA) and placed on one of the forearms. Due to the small number of animals in each group, no statistical analysis was performed.

[0265] Experiments have shown that: Toxicological studies during the first 3 weeks in Wistar rats Following three consecutive weekly intravenous injections, molecule-1 at three dose levels resulted in premature euthanasia in 3 out of 16 rats administered 60 mg / kg due to clinical signs (ventral pitting, piloerection, ptosis), and at the end of the study (day 21), a -25% decrease in body weight in males and a -12% decrease in body weight in females compared to the control. Figure 13 Following each weekly dosing, a transient reduction in body weight (BW) was observed at 60 mg / kg, with the lowest values ​​at 3–4 days post-injection being up to -7% in males (up to -27% relative to control) and -10% in females (up to -15% relative to control). At 30 mg / kg, body weight decreased by -10% to -11% (compared to control) in both sexes on day 21. During the 21-day observation period, weight gain was virtually nonexistent in both sexes at 60 mg / kg and almost 50% reduced at 30 mg / kg compared to control. Food intake decreased by 33–37% in a dose-dependent manner at 60 mg / kg and by 16–19% at 30 mg / kg.

[0266] On day 22, one week after the last dose (day 15), clinicopathological measurements showed at most a moderate decrease in erythrocytes, hemoglobin, and hematocrit (all parameters at most -29% at 60 mg / kg and at most -9% at 30 mg / kg), with an increase in reticulocytes (up to 90%) at the intermediate dose indicating regeneration, and no change in reticulocytes at the high dose. At 60 mg / kg, leukocytes, lymphocytes, and eosinophils appeared to decrease, and at 30 mg / kg, a partial decrease (eosinophils in males and lymphocytes in females). At both intermediate and high doses, platelets increased (up to 60%), while neutrophil counts showed a trend toward decrease (not statistically significant). Choosing day 22 for hematological analysis may not be optimal regarding hematopoietic regeneration capacity in rats. Regarding the hematologic toxicities described in rodents, such as neutropenia, lymphopenia, and thrombocytopenia (Verschraegen et al., 2000), these effects likely partially recovered 7 days after the last dose of molecule 1. As for clinical blood chemistry, total protein (-14%) and albumin (-12%) decreased only at 60 mg / kg. The latter effect is most likely related to general catabolism in the absence of any weight gain over 3 weeks.

[0267] Microscopic examination of organs collected on day 22 revealed adverse findings in the hemolymphatic system (reduced cellular composition and / or increased unicellular necrosis in the periarterial lymphatic sheaths (PALS) of the thymus, bone marrow (BM), and spleen, and lymph nodes), the gastrointestinal tract (increased unicellular necrosis in crypt regions), and reproductive organs (degeneration of oocytes, granulosa cells, and seminiferous tubules), reflecting patterns associated with antimitotic cytotoxic agents. In the thymus, moderate to significant loss of the corticomedullary boundary was observed in the most severely affected rats. Additionally, particularly in individual rats in the 60 mg / kg group, minor findings included reduced primary trabeculae in the bone / bone marrow, woven bone formation or fibrosis, and skin ulceration. Woven bone formation and fibrosis can be seen as compensatory attempts to compensate for the disrupted bone formation. In the four male rats in the 60 mg / kg group, the accompanying ulcerated skin wounds were likely due to opportunistic infections (coccal colonies were present in microscopic sections) of skin changes (scratches or bites) that typically occur under immunosuppression. Loss of germinal centers in the lymph nodes of most rats was observed starting at low and higher doses of 10 mg / kg, indicating a high sensitivity of this specific B-cell compartment in rats to the antimitotic activity of molecule-1. Other observations at 60 mg / kg included minimal extramedullary hematopoiesis in the liver and adrenal glands, considered an adaptive phenomenon due to bone marrow suppression. Prematurely euthanized females from the main 60 mg / kg group (on day 14 after two doses) exhibited multifocal, moderate erosion in the cecum, likely contributing to their poor clinical condition. Other findings in these rats were comparable to those in the rest of the group. No histopathological examination was performed on the two prematurely euthanized rats from the satellite TK group (female on day 5, male on day 18).

[0268] Microscopic evaluation of rat dorsal root ganglia (neck, thorax, lumbar) and peripheral nerves (including optic nerve, tibial nerve, fibular nerve and sciatic nerve) did not reveal any abnormalities.

[0269] Toxicokinetics assessment Within the tested dose range, overall plasma exposure for total antibody, conjugated (eciletecan), and unconjugated (released eciletecan) increased proportionally with increasing dose, with ADC accumulation observed in the 10 and 30 mg / kg treatment groups, but not in the 60 mg / kg group. Maximum plasma concentrations of unconjugated eciletecan at very low plasma levels were observed between 0.5 and 4 hours, with a half-life similar to that of ADCs (approximately 2–3 days), indicating a rate-limiting process of ADC formation. No significant sex-related differences in plasma exposure were observed.

[0270] vi. A lead 3-week toxicology study in cynomolgus monkeys, accompanied by CV function evaluation. Molecular 1 was tolerated at three dose levels in cynomolgus monkeys administered once weekly for three consecutive weeks. A fourth, higher dose of 32 mg / kg of molecule 1 administered to a single male monkey was intolerable after two administrations (day 1 and day 8) and required sacrifice on day 14. Therefore, in this study, the dose of 32 mg / kg was considered to exceed the mean time to treatment (MTD).

[0271] Up to the mean time to dose (MTD), the monkeys exhibited dose-dependent gastrointestinal clinical signs associated with weight loss (i.e., episodes of soft stools and / or diarrhea). Mild episodes of soft stools and / or diarrhea were observed in both animals and in females at an MTD of 16 mg / kg. This was associated with a gradual decrease in body weight (BW) of -11% (day 14) to -16% (day 21) over time in females but not males at 16 mg / kg. A mild decrease in BW was observed in both sexes at 8 mg / kg and in male monkeys at 4 mg / kg. Hematological investigations in the monkeys over time (…) Figure 14 The results showed minimal to slight reductions in erythrocytes, hemoglobin, and hematocrit at all dose levels (up to -27% relative to baseline in both sexes at 16 mg / kg), accompanied by limited dose-response relationships. Regenerative responses (i.e., up to a 5-fold increase in reticulocyte counts) were observed at 4 and 8 mg / kg, while at 16 mg / kg, moderate to severe reductions were noted for the same parameters (up to approximately -95% at day 8). At 16 mg / kg, neutrophil counts transiently decreased in male monkeys at days 8 and 15 (<2.010). 3 ( / μL), but still higher than the lower limit of the normal reference value (=1.10x10). 3 / μL, average 5.77 ± 3.57 x10 3 / μL, as reported by Park et al., 2016).

[0272] At 16 mg / kg, histopathology was limited to changes in the lymphoid system (hypoplasticity in lymph nodes and thymus). Mild effects in the thymus in all monkeys treated at 8 mg / kg and 4 mg / kg may be confounded by secondary stress-related effects. No histopathological changes were observed in the GI pathway.

[0273] At any dose tested, including in male monkeys exceeding the MTD (post-mortem on day 14), microscopic evaluation of the peripheral nerves (including the optic nerve, tibial nerve, fibular nerve, and sciatic nerve) did not reveal any findings relevant to the test items.

[0274] Cardiovascular function Until MTD did not measure any changes in heart rate (HR), ECG (QT / HR-corrected QT), systolic blood pressure, diastolic blood pressure, mean blood pressure, respiratory rate, or body temperature.

[0275] Toxicokinetics assessment Within the tested dose range, overall plasma exposure for total antibody, conjugated (eciletecan), and unconjugated (released eciletecan) increased proportionally with increasing dose, without any analyte accumulation. A decrease in exposure was observed only in a single male monkey after repeated administration of 4 mg / kg, indicating potential ADA formation in that subject. Maximum plasma concentrations of unconjugated eciletecan at very low levels were typically observed after 6 hours, with a half-life similar to that of ADCs (approximately 2–3 days), indicating a rate-limiting process of ADC formation. No significant sex-related differences in plasma exposure were observed.

[0276] Discussion of experimental findings Despite improved survival, acute neuropathic pain is a common and critical dose-limiting adverse event (AE) associated with peripheral neuropathy, observed with GD2 immunotherapy using currently marketed anti-GD2 antibodies (datuximab, nalcitumab) in the treatment of patients with neuroblastoma and osteosarcoma. In a lead toxicity / TK study using elegans-based ADC molecule 1 administered once weekly (3 times) in rats and cynomolgus monkeys, no PNS impairment was observed at day 22, and no indication of pain was noted in their behavior. Even after a single dose, molecule 1 resulted in potent antitumor activity in a GD2-expressing xenograft mouse model. These nonclinical data collectively demonstrate a positive benefit-risk ratio, likely indicating improved clinical prospects for the treatment of GD2-expressing tumors in pediatric and adult patients relative to current GD2 immunotherapy. Generally, the absence of aberrant signs of defensive behavior such as overreaction, anxiety, or agitation during treatment may indicate mechanical aberrant pain. Furthermore, no signs of pain, such as reduced motor activity, upright posture, or exploration, were observed in their caged behavior. Additionally, trained monkeys seated in chairs received 30 minutes of intravenous infusion without problems, and subsequent measurements of CV function that might respond to painful stimuli, such as tachycardia or elevated blood pressure (albeit nonspecific), appeared to remain normal in the monkeys one hour later.

[0277] In contrast, dartuximab beta infusion for 4 hours / day for 10 consecutive days (30 and 100 mg / m²) 2In a toxicity study of cynomolgus monkeys (days), histopathological changes were observed in the peripheral nerves and visceral innervation of organs at day 15 (EMA / 263814 / 2017, 2017). These observations, along with clinical signs (e.g., reduced activity) in these monkeys, were interpreted as signs most likely associated with acute pain induction due to a test item targeting GD2 within the peripheral nervous system. Some patients with melanoma have developed sensorimotor demyelinating polyneuropathy after treatment with anti-GD2 mAb (14G2a; ch14.18 derived from this mouse IgG2a isotype) (Yuki et al., 1997). Toxicological studies have not been conducted in monkeys or other non-rodent animals with nalcitumab (FDA BLA #761171, 2020) or dartuximab (Unituxin®, FDA BLA #125516, 2014).

[0278] In the DRF toxicity study, no microscopic changes were observed in several examined peripheral nerves (sciatic, tibial, fibular, and optic nerves, and DRG at different levels in rats) in monkeys and rats after administration of repeat molecule 1, confirming adequate plasma exposure to the ADC. Clinical signs in monkeys were limited to an occasional occurrence of mild soft stools and diarrhea only at the MTD of 16 mg / kg, and a decrease in body weight (BW) in females (-16%) but not males. A dose-dependent decrease in BW and a slight decrease in food intake were observed primarily in rats. Since 3 out of 16 rats were in poor condition without an increase in BW at 60 mg / kg, the high dose in this DRF rat study was considered to exceed the MTD. Based on clinical and histopathological examination, monkeys and rats showed hemolymphatic and GI tract effects similar to eczema toxicity (Verschraegen et al., 2000; Rowinsky 2005), which were attributed to relatively low plasma levels of unconjugated eczema released from the ADC over a week. Neutropenia is considered a major dose-limiting toxicity of eczemab mesylate (as observed in cancer patients and animals), along with other hematologic toxicities (such as anemia, lymphopenia, and thrombocytopenia) and GI effects.

[0279] As detailed in Experiment 7, no signs of immunogenicity were observed in rats and monkeys after repeated administration of molecule 1 over three weeks (except for one monkey with a low dose), as judged by the TK profile and the absence of abnormal toxicity, which showed normal dose-proportioning and regular TK curves. The absence of immunogenicity is likely explained by the strong immunosuppressive effects induced by molecule 1 (e.g., absence of germinal centers, major B cell compartments). Other non-hematologic toxicities observed with molecule 1, such as antiproliferative changes in reproductive organs / tissues in female and male rats, can be attributed to the antimitotic effects of eczema (Verschraegen et al., 2000; Rowinsky 2005).

[0280] By altering the concept of tumor killing by replacing it with targeted delivery of a non-neurotoxic payload and eliminating any Ab Fc-effector functionality, PNS damage was prevented in both tested animal species. A significant advantage of this consistent result is that molecule 1 may at least avoid or reduce potential PNS damage in patients.

[0281] In some embodiments of the invention, the treatment method describes the PNS-forgiving activity of molecule 1, which enables the prevention of neurological disorders of the eye (e.g., mydriasis, blurred vision, unequal pupils, or photophobia).

[0282] Experiment 8: ADCs for the treatment of osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma The following data confirm the efficacy of GD2-ADC in the treatment of solid tumors. In the selected implementation method, such as... Figure 18-21 As shown, the anti-GD2 [hu14.18-IgG1.4(K322A)-delK (MBE-91)] antibody (the ADC referred to as "Molecular 1") conjugated with a small molecule toxicity payload from the eczema topoisomerase I inhibitor class has demonstrated efficacy in cancer treatment, including (but not limited to) neuroblastoma, osteosarcoma, glioma, and soft tissue sarcoma. Molecular 1 has shown strong antitumor activity in a group of patient-derived xenografts (PDXs) from the aforementioned cancer types, indicating its antitumor efficacy in patients with neuroblastoma, osteosarcoma, glioma, and soft tissue sarcoma.

[0283] Legend for Experiment 8 Figure 18 In a group of nine different patient-derived xenografts from neuroblastoma patients, a single dose of 10.0 mg / kg molecule 1 resulted in strong and significant antitumor activity in all tested PDX models.

[0284] Figure 19In a group of 12 different patient-derived xenografts from osteosarcoma patients, a single dose of 10.0 mg / kg molecule 1 resulted in significant antitumor activity in 9 PDX models.

[0285] Figure 20 In a group of nine different patient-derived xenografts from glioma patients, a single dose of 10.0 mg / kg molecule 1 resulted in strong and significant antitumor activity in all tested soft tissue sarcoma PDX.

[0286] Figure 21 In a group of seven different patient-derived xenografts from soft tissue sarcoma, a single dose of 10.0 mg / kg molecule 1 resulted in significant antitumor activity in all PDX models tested for soft tissue sarcoma.

Claims

1. A method for treating solid tumor cancer, the method comprising administering an antibody-drug conjugate (ADC) to a patient in need, wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO: 1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation.

2. The method of claim 1, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

3. The method according to claim 1, wherein the growth inhibitor is eczema.

4. The method according to claim 1, wherein the solid tumor cancer is selected from: osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

5. An antibody-drug conjugate (ADC) for use in the treatment of solid tumor cancer, wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO: 1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation.

6. The ADC for use according to claim 5, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

7. The ADC for use according to any one of claims 5 or 6, wherein the growth inhibitor is eczema.

8. The ADC for use according to any one of claims 5 to 7, wherein the solid tumor cancer is selected from osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

9. Use of an antibody-drug conjugate (ADC) for the manufacture of an agent for the treatment of solid tumor cancer, wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO: 1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a mutation that reduces complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein the mutation results in an absolute reduction in complement fixation.

10. The use according to claim 9, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

11. The use according to claim 9, wherein the growth inhibitor is eczema.

12. The use according to claim 9, wherein the solid tumor cancer is selected from: osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

13. A pharmaceutical composition for treating solid tumor cancer, the pharmaceutical composition comprising an antibody-drug conjugate (ADC), wherein the ADC comprises a growth inhibitor and / or antiproliferator linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO: 1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation.

14. The pharmaceutical composition of claim 13, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

15. The pharmaceutical composition according to claim 13, wherein the growth inhibitor is eczema.

16. The pharmaceutical composition according to claim 13, wherein the solid tumor cancer is selected from: osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

17. Use of an antibody-drug conjugate (ADC) for the treatment of solid tumor cancer, wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO: 1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO: 4, and an Fc region having a mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation.

18. The use according to claim 17, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

19. The use according to claim 17, wherein the growth inhibitor is eczema.

20. The use according to claim 17, wherein the solid tumor cancer is selected from: osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

21. Use of a pharmaceutical composition comprising an antibody-drug conjugate (ADC) for the treatment of solid tumor cancer, wherein the ADC comprises a growth inhibitor and / or antiproliferative agent linked to an antibody via a linker, wherein the antibody comprises a light chain variable region of an anti-GD2 antibody of SEQ ID NO:1, a heavy chain variable region of an anti-GD2 antibody of SEQ ID NO:4, and an Fc region having a mutation, said mutation reducing complement fixation relative to antibody-dependent cell-mediated cytotoxicity, wherein said mutation results in an absolute reduction in complement fixation.

22. The use according to claim 21, wherein the solid tumor cancer exhibits high levels of cell surface GD2 expression.

23. The use according to claim 21, wherein the growth inhibitor is eczema.

24. The use according to claim 21, wherein the solid tumor cancer is selected from: osteosarcoma, glioma, neuroblastoma, and soft tissue sarcoma.

Images