Antibody conjugates for targeting Trop-2 expressing tumors

Abstract

The present invention relates to antibody conjugates which are particularly suitable for targeting Trop-2 expressing cells, in particular tumor cells. The antibody conjugate according to the present invention has structure (1): wherein AB is an antibody capable of targeting a tumor expressing Trop-2; l is a joint connecting Z to D; z is a linking group; l6 is-GlcNAc (Fuc) w-(G) j-S-(L7) w '-wherein G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L7 is-N (H) C (O) CH2-,-N (H) C (O) CF2-or-CH2-; d is Iproxetecan; b is 0 or 1; x is 1 or 2; and y is 1, 2, 3 or 4. The invention further relates to a method for preparing said antibody conjugate of structure (1) and to the use of said antibody conjugate of structure (1).

Description

Technical Field

[0001] The present invention belongs to the field of bioconjugation. More specifically, the present invention relates to antibody drug conjugates for targeted therapy of patients with cancer, especially tumors expressing Trop-2. Background Art

[0002] A promising approach to targeted tumor therapy requires conjugating a large number (2 to 8) of highly toxic payloads to monoclonal antibodies to produce antibody-drug conjugates (ADCs). ADCs are well known in the art, such as by Chari et al., Angew. Chem. Int. Ed. 2014, 53, 3796 and Beck et al., Nat. Rev. Drug Discov. 2017, 16, 315–37. Mechanistically, the antibody is designed to bind to tumor-associated receptors that are overexpressed compared to healthy tissues with high specificity. ADCs are thought to be internalized into tumor cells after binding to the receptors, and then release the toxic payload when the antibody and / or linker is degraded in the lysosome.

[0003] Current ADCs are usually prepared by various conjugation techniques (summarized in Figure 1In the present invention, the conjugation technology is mainly based on the conjugation of maleimide to cysteine ​​side chains or activated esters to lysine side chains. In order to produce ADCs based on native cysteine ​​that naturally participates in disulfide bonds, the sulfhydryl groups in the side chains can be released by subjecting the antibody to a suitable reducing agent (such as TCEP or DTT) and then treating it with a maleimide-functionalized linker-drug. In the event that only a total of eight free sulfhydryl groups are not fully alkylated, the resulting ADC usually consists of a mixture of positional isomers. Alternatively, in order to produce site-specific ADCs, antibodies can be produced by mutating one or more amino acids at defined positions in the sequence to cysteine, the side chain of which can be selectively released for alkylation by a reduction-oxidation sequence. Commonly used cysteines for site-specific conjugation are LC-41C (light chain 41C), HC-41C (heavy chain 41C), LC-80C, HC-118C, HC-265C, HC-140C, LC-149C, LC-124C, LC-180C, HC-190C, HC-160C, LC-183C, HC-290C, LC-205C, HC-220C, HC-239C, HC-442C. Additional cysteines may also be inserted into the sequence, such as HC-i239C. In addition to maleimide as an alkylating agent, reactions of cysteine ​​side chains with haloacetamides or vinylbenzene derivatives have also been reported. In addition to reacting with natural amino acid side chains, specific non-natural (non-canonical) amino acids can also be designed into the amino acid sequence of the antibody to provide unique handles for chemical conjugation, such as ketones, acetylenes, azides, cycloalkynes or cycloalkenes to react with oximes, azides, alkynes or tetrazines, respectively. However, the disadvantage of the latter approach is that the natural sequence of the antibody must be redesigned, which, in addition to being time-consuming and expensive, may also lead to instability issues.

[0004] Conjugation through oxidation-linking sequences, through glycans is known in the art and has been described, for example, by Hamann et al. (Bioconjugate Chem. 2002, 13, 47-58). Chemoenzymatic conjugation through glycans is known in the art and has been described by Boons et al., Angew. Chem. Int. Ed. 2014, 53, 7179 using sialyltransferases, and by Zhu et al., mAbs 2014, 6, 1 and Cook et al., Bioconjugate Chem. 2016, 27, 1789 using mutant galactosyltransferases.

[0005] Chemoenzymatic conjugation of glycans (including first trimming the glycans) is known in the art and has been described by van Geel et al., Bioconjugate Chem. 2015, 26, 2233 and schematically depicted in Figure 2In brief, monoclonal antibodies are treated with endoglycosidases to trim the glycans to the core GlcNAc (directly attached to Asn-297), and then the azido-modified sugar is transferred under the action of glycosyltransferases. The various structures of UDP-azido sugars are shown in Figure 3 As shown. A particularly suitable combination involves the transfer of GalNAz 2b (2-azidoacetyl-N-galactosamine) under the action of a mutant galactosyltransferase GalT (Y289L), as disclosed in WO 2007 / 095506, EP 2911699 B1 and van Geel et al. Another powerful combination requires 6-azidoGalNAc 2d with a native GalNAc transferase, as disclosed in PCT / EP2016 / 059194. Another useful combination requires GlcNAz with α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT1), as disclosed in WO2021 / 248048.

[0006] Various cyclooctynes ​​for metal-free click chemistry are known in the art ( Figure 4 ). In particular, various cyclooctynes ​​such as DIBO (I), DBCO / DIBAC (J), s-DIBO (K), BCN (L), and TMTHSI (T) are often used for conjugation with azides.

[0007] The payload of ADC is usually a highly cytotoxic molecule with an IC 50 Values ​​are in the low nanomolar or picomolar range, particularly for low to medium molecular weight compounds (e.g., about 200 to about 2500 Da). Examples of cytotoxic classes suitable for ADCs include anthracyclines, camptothecins, taxanes, tubulysins, enediynes, inhibitory peptides, amanitin, duocarmycins, maytansinoids, auristatins, eribulins, hemiasters, BCL-XL inhibitors, KSP inhibitors, TLR agonists, indoline and benzodiazepines. Pyrrolobenzodiazepine Dimer (PBD) and its analogs or prodrugs. Figure 5 Depicted is a representative panel of cytotoxic molecules and / or synthetic derivatives or prodrugs thereof having suitable attachment points for conjugation to monoclonal antibodies.

[0008] Specific examples of anthracyclines suitable for use in ADCs include, but are not limited to, doxorubicin, daunorubicin, neomycin, and PNU-159,682.

[0009] Specific examples of camptothecins suitable for use in ADCs include, but are not limited to, SN-38, exotecan, exotecan-S, topotecan, silatecan, costitecan, lortotecan, gimatecan, belotecan, rubitecan, AMDCPT, G-AMDCPT and other synthetic camptothecins, whose structures are as shown in Figure 6 As shown. EP0296597, WO2019 / 236954, WO2020 / 200880, WO2020 / 219287, CN113816969, CN113710277 and US20180200273 disclose a variety of novel camptothecins.

[0010] Specific examples of enediynes suitable for use in ADCs include, but are not limited to, calicheamicin, esperamicin, shishijimicin, and namenamicin, as well as other enediynes summarized in Galm et al., Chem. Rev. 2005, 105, 739-758.

[0011] Specific examples of auristatins suitable for use in ADCs include, but are not limited to, MMAD, MMAE, MMAF, and PF-06380101, and other auristatins summarized in Maderna et al., Mol. Pharmaceutics 2015, 12, 1798-1812.

[0012] Proteins that play a role in breast cancer growth, differentiation, invasion and / or metastasis can influence the biological progression of the tumor and, therefore, can provide important prognostic information. One such candidate is Trop-2 (GA733-1, EGP-1), a 45 kDa monomeric transmembrane glycoprotein that belongs to the TACSTD gene family, specifically TACSTD2, which is expressed in human epithelial cells at different stages of differentiation. Overexpression of Trop-2 has been shown to be necessary and sufficient to stimulate tumor growth and is associated with an overall poor prognosis. Expression of Trop-2 is associated with a poor prognosis in a variety of human cancers, including oral, pancreatic, gastric, ovarian, colorectal, breast, and lung cancers. For example, overexpression of Trop-2 was observed in 55% of pancreatic cancer patients studied and was positively correlated with metastasis, tumor grade, and poor progression-free survival in patients who underwent radical surgery. Similarly, in gastric cancer, 56% of patients may show overexpression of Trop-2 on their tumors, which is again associated with shorter disease-free survival and worse prognosis in patients with lymph node involvement of Trop-2-positive tumor cells.

[0013] Given these characteristics and the fact that Trop-2 is associated with so many refractory cancers, Trop-2 is an attractive target for therapeutic intervention. However, it must be considered that Trop-2 is also expressed in some normal tissues, although usually at a much lower intensity compared to tumor tissues and often in tissue areas with restricted vascular access.

[0014] Several monoclonal antibodies against Trop-2 have been established. Some anti-Trop-2 monoclonal antibodies, such as 77220, are commercially available as reagents. Some of these established anti-Trop-2 monoclonal antibodies are being studied for the treatment of cancer. Most of the anti-Trop-2 monoclonal antibodies currently available on the market, such as T16, are produced using myeloma cell lines (such as NS-1 or SP2-1) as fusion partners, and T16 retains the expression of the parent immunoglobulin light chain. This results in these antibodies being actually a heterogeneous mixture of antibodies, in which one, two, or none of the two light chains are directly involved in Trop-2 recognition.

[0015] WO 1997 / 14796 describes monoclonal antibody BR110, which is known to bind to Trop-2 on the cell surface and internalize in the cell. Patent applications WO 2003 / 074566, US2004 / 001825, US2007 / 212350 and US 2008 / 131363 and patents US10,179,171B2 and EP3483183B1 teach RS7 antibodies and their use in the treatment and diagnosis of tumors. These applications further relate to humanized, human and chimeric RS7 antigen binding proteins (hRS7, sacituzumab), and the use of such binding proteins in diagnosis and treatment. Anti-Trop-2 monoclonal antibody AR47A6.4.2 is disclosed in WO 2007 / 095748, and AR52A301.5 is disclosed in WO 2007 / 095749, both of which are antibodies that specifically damage cancer cells expressing Trop-2 as target cells to exhibit effects. Patent application WO 2008 / 144891 teaches a humanized form of AR47A6.4.2 as an anti-Trop-2 monoclonal antibody for treating tumors. Patent application WO 2011 / 155579 (Sapporo) teaches a humanized monoclonal antibody AR47A6.4.2 or an antibody fragment thereof that binds to the extracellular region of human Trop-2 with high affinity and exhibits high ADCC activity and high anti-tumor activity. Patent applications WO 2013 / 077458 and US9427464B2 (LivTech / Chiome) teach anti-human Trop-2 antibodies with anti-tumor activity, in particular humanized antibodies including Huk5-70-2, particularly with in vivo anti-tumor activity. Patent applications WO 2013 / 068946 and US8,871,908B2 (Rinat / Pfizer) teach antibodies 7E6 and humanized h7E6 that specifically bind to trophoblast cell surface antigen 2 (Trop-2). The present invention also relates to therapeutic methods using these antibody conjugates to treat conditions (e.g., cancer) associated with Trop-2 expression, such as colon cancer, esophageal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, or pancreatic cancer. Patent US8,715,662 (Oncoxx) teaches antibodies 2G10 that specifically bind to TROP-2.

[0016] ADCs targeting Trop-2 are known in the art and are in various stages of clinical development. IMMU-132 is an ADC derived from the humanized antibody hRS7 / certolizumab pegol, which is conjugated to the camptothecin analog SN-38 via an acid-cleavable linker CL2A, as disclosed in WO 2015 / 012904, and is currently in late clinical development for the treatment of a range of clinical indications, including (triple-negative) breast cancer, small cell lung cancer, and pancreatic cancer. DS-1062a (datopotamab-deruxtecan) is an ADC derived from the humanized antibody hTINA / datopotamab, which is conjugated to the camptothecin analog DXd through a protease-sensitive GGFG cleavable linker, as disclosed in WO 2015 / 098099, US11,008,398B2 and EP3088419B1, and is under clinical evaluation for the treatment of solid tumors. Third, PF-06664178 is an ADC derived from the monoclonal antibody RN926, which is site-specifically conjugated to the auristatin analog PF-06380101 under the action of microbial transglutaminase. PF-06664178 was evaluated in a Phase I clinical study in patients with advanced or metastatic solid tumors, but the ADC showed toxicity at high dose levels and only modest antitumor activity, so development was halted. Summary of the invention

[0017] The inventors have developed antibody conjugates that are very suitable for targeting cells, in particular tumors, expressing Trop-2. Therefore, the antibody conjugates according to the invention are very suitable for treating Trop-2 positive cancers, in particular breast cancer, colorectal cancer, pancreatic cancer, lung cancer, bladder cancer, head and neck cancer, ovarian cancer or esophageal cancer.

[0018] In a first aspect, the present invention relates to an antibody conjugate. Related thereto, in a second aspect, the present invention relates to a method for preparing an antibody conjugate according to the present invention. In a third aspect, the present invention relates to a method for targeting cells expressing Trop-2. Related thereto is a first medical use of an antibody conjugate according to the present invention, and a second medical use for treating cancer. In a last aspect, the present invention relates to the use of the conjugation mode for increasing the therapeutic index of the antibody conjugate in the treatment of tumors expressing Trop-2. DETAILED DESCRIPTION

[0019] definition

[0020] The verb "to comprise" and its conjugations as used in this specification and claims are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0021] Furthermore, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0022] A joint is defined herein as a portion that connects (covalently links) two or more elements of a compound. A joint may include one or more spacer moieties. A spacer moiety is defined herein as a portion that spaces (i.e., provides a distance between) two (or more) portions of a joint and covalently links them together. A joint may be, for example, a portion of a joint-construct, a joint-conjugate, a joint-payload (e.g., a joint-drug) or an antibody conjugate, as defined below.

[0023] A "hydrophilic group" or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that impart improved polarity to the molecule to which it is attached and thus provide improved water solubility. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonate groups, phosphate groups, acylsulfonamide groups or carbamoylsulfonamide groups. In addition to higher solubility, other effects of the hydrophilic group include improved click conjugation efficiency and, once incorporated into the antibody-drug conjugate: reduced aggregation, improved pharmacokinetics, resulting in higher efficacy and in vivo tolerability.

[0024] The term "salt thereof" means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, but this is not necessary for salts that are not intended to be administered to a patient. For example, in a salt of a compound, the compound can be protonated by an inorganic or organic acid to form a cation, with a conjugated base of the inorganic or organic acid as the anionic component of the salt. The term "pharmaceutically acceptable salt" means an acceptable counterion for administration to a patient, such as a mammal (a counterion with acceptable mammalian safety for a given dosage regimen). Such salts can be derived from a pharmaceutically acceptable inorganic or organic base and a pharmaceutically acceptable inorganic or organic acid. "Pharmaceutically acceptable" salts refer to pharmaceutically acceptable salts of compounds which are derived from various organic and inorganic counterions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

[0025] The term "enediyne" or "enediyne antibiotic" or "enediyne-containing cytotoxin" refers to any cytotoxin known in the art that is characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule, and includes neocarzinostatin (NCS), C-1027, kedarcidin (KED), maduropeptin (MDP), N1999A2, sporolide (SPO), cyanosporaside (CYA and CYN), and fijiolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamicin, shishijimicin, and uncialamycin (UCM).

[0026] The term "alkylamino sugar" as used herein refers to a tetrahydropyranyl moiety attached to an alcohol functional group via its 2-position, thereby forming an acetal functional group, and further substituted with (at least) one N-alkylamino group at position 3, 4 or 5. "N-alkylamino group" herein refers to an amino group having one methyl, ethyl or 2-propyl group.

[0027] The term "click probe" refers to a functional part that can undergo a click reaction, i.e. two compatible click probes undergo a click reaction with each other such that they are covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of the present invention, the click probe Q in the compound according to the invention is capable of reacting with the click probe F on the (modified) protein such that when the click reaction occurs, a conjugate is formed in which the protein is conjugated to the compound according to the invention. Here, F and Q are compatible click probes.

[0028] "Acylsulfonamide moiety" is defined herein as a sulfonamide moiety (H2NSO2NH2) that is N-acylated or N-carbamylated at one end of the molecule and N-alkylated (single or double) at the other end of the molecule. In the context of the present invention, particularly in the examples, this group is also referred to as "HS".

[0029] A "domain" can be any region of a protein, typically defined based on sequence homology and typically associated with a specific structural or functional entity. As used herein, the term domain refers to a single Ig-like domain (e.g., a "V domain") or a group of consecutive domains (such as a "C2 1-2 type domain").

[0030] A "coding sequence" or a sequence "encoding" an expression product such as an RNA, polypeptide, protein or enzyme is a nucleotide sequence that, when expressed, results in the production of the RNA, polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of the polypeptide, protein or enzyme. The coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0031] The term "gene" means a DNA sequence encoding or corresponding to a specific amino acid sequence, which includes all or part of one or more proteins or enzymes, and the DNA sequence may include or may not include a regulatory DNA sequence, such as a promoter sequence, which, for example, determines the conditions for gene expression. Some genes are not structural genes and can be transcribed into RNA from DNA, but are not translated into amino acid sequences. Other genes may serve as regulators of structural genes or regulators of DNA transcription. Specifically, the term gene may mean a genomic sequence encoding a protein, i.e. a sequence comprising a regulator, a promoter, an intron, and an exon sequence.

[0032] The term "glycoprotein" is used herein in its normal scientific sense and refers to a protein comprising one or more monosaccharide or oligosaccharide chains ("glycans") covalently bonded to the protein. The glycan may be attached to a hydroxyl group on the protein (O-linked glycans), such as to the hydroxyl groups of serine, threonine, tyrosine, hydroxylysine or hydroxyproline, or to an amide functional group on the protein (N-glycoproteins), such as asparagine or arginine, or to a carbon on the protein (C-glycoproteins), such as tryptophan. A glycoprotein may comprise more than one glycan, may comprise a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may comprise a combination of N-linked, O-linked and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and therefore qualify as glycoproteins. Examples of glycoproteins include PSMA (prostate specific membrane antigen), CAL (Candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0033] The term "glycan" is used herein with its normal scientific meaning and refers to a monosaccharide or oligosaccharide chain connected to a protein. Therefore, the term glycan refers to the carbohydrate portion of a glycoprotein. A glycan is connected to a protein via the C-1 carbon of a sugar, which may not be further substituted (monosaccharide), or may be further substituted (oligosaccharide) at one or more of its hydroxyl groups. Naturally occurring glycans typically contain 1 to about 10 sugar moieties. However, when a longer sugar chain is connected to a protein, the sugar chain is also considered to be a glycan herein. The glycan of a glycoprotein may be a monosaccharide. Typically, the monosaccharide glycan of a glycoprotein is composed of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to a protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In oligosaccharides, the sugar directly attached to a protein is referred to as a core sugar. In oligosaccharides, a sugar that is not directly attached to a protein but is attached to at least two other sugars is referred to as an endosugar. In oligosaccharides, it is not directly connected to the protein but connected to a single other sugar, that is, a sugar that does not carry other sugar substituents on one or more of its other hydroxyl groups, which is called a terminal sugar. For the avoidance of doubt, multiple terminal sugars can be present in the oligosaccharides of glycoproteins, but there is only one core sugar. Polysaccharides can be O-linked polysaccharides, N-linked polysaccharides or C-linked polysaccharides. In O-linked polysaccharides, monosaccharides or oligosaccharides polysaccharides are usually bonded to the O atoms in the amino acids of proteins via the hydroxyl groups of serine (Ser) or threonine (Thr). In N-linked polysaccharides, monosaccharides or oligosaccharides polysaccharides are bonded to proteins via the N atoms in the amino acids of proteins, usually via the amide nitrogen in the side chains of asparagine (Asn) or arginine (Arg). In C-linked polysaccharides, monosaccharides or oligosaccharides polysaccharides are bonded to the C atoms in the amino acids of proteins, usually with the C atoms of tryptophan (Trp).

[0034] The term "antibody" (AB) is used in this article with its normal scientific meaning.Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens.Antibodies are an example of glycoproteins.The term antibody herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies.The term "antibody" is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens.The term "antibody" is intended to include complete antibodies, but also includes antibody fragments, such as antibody Fab fragments, F(ab')2, Fv fragments or Fc fragments from cleaved antibodies, scFv-Fc fragments, mini antibodies, double antibodies, or scFv.In addition, the term includes genetically engineered antibodies and antibody derivatives.Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0035] Antibodies can be natural or conventional antibodies, in which two heavy chains are connected to each other by disulfide bonds and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains: λ (l) and κ (k). The light chain includes two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant regions of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and is composed of the variable portion of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass or allotype (e.g., human G1m1, G1m2, G m3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of immunoglobulin molecules. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2 or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. The antibody can be engineered in the Fc domain to enhance or eliminate binding to the Fc-γ receptor, as summarized by Saunders et al. Front. Immunol. 2019, 10, doi: 10.3389 / fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131–141. For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. Elimination of binding to Fc-γ receptors can also be achieved by mutating the N297 amino acid to any other amino acid except asparagine, by mutating the T299 amino acid to any other amino acid except threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies using, for example, PNGase F or endoglycosidases. The immunoglobulins can be derived from any species, including human, mouse or rabbit sources. Each chain contains different sequence domains.

[0036] The percentage of "sequence identity" is determined by comparing two optimally aligned sequences within a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). A sequence that is "at least 85% identical to a reference sequence" is one that has 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its entire length to the entire length of the reference sequence.

[0037] The term "CDR" refers to complementarity determining region: the specificity of an antibody lies in the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues mainly from the hypervariable regions or complementary determining regions (CDRs). Sometimes, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and therefore the binding site. The complementary determining region or CDR therefore refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site includes six CDRs, which contain a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0038] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule that is directed against a single amino acid sequence of a specific antigen, and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0039] The term "chimeric antibody" refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies in its broadest sense. In one embodiment, a chimeric antibody comprises a VH domain and a VL domain derived from an antibody of a non-human animal, which are combined with a CH domain and a CL domain of another antibody (in one embodiment, a human antibody). As non-human animals, any animal such as mice, rats, hamsters, rabbits, etc. can be used. A chimeric antibody can also refer to a multispecific antibody that is specific for at least two different antigens.

[0040] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified, for example, in the framework regions of the VH and VL domains to replace certain amino acids in order to avoid or minimize human immune responses. The constant domains of humanized antibodies are most often human CH and CL domains. A "fragment" of a (conventional) antibody comprises a portion of a complete antibody, in particular the antigen binding region or variable region of a complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed by antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A representative set of reactive groups (F) are shown, which, when present in a biomolecule, will form linker groups Z (1a-1h) upon reaction with reactive group Q. Functional group F may exist naturally or may be artificially introduced (engineered) into a biomolecule at any selected position.

[0042] Figure 2 Schematically shows how to obtain antibody conjugates from any monoclonal antibody by a two-stage process. In the first stage, azido-modified UPD-Gal or UDP-GalNAc can be attached to the monoclonal antibody in a one-pot process involving (a) trimming polysaccharides (to core GlcNAc) by endoglycosidases and (b) attaching azido-sugars under the action of glycosyltransferases (galactosyltransferases or mutants thereof or GalNAc-transferases), thereby generating β-glycosidic 1-4 bonds between azido-modified GalNAc and GlcNAc. In the second stage, the azido-modified antibody is reacted with appropriately functionalized cyclooctynes ​​to produce antibody conjugates.

[0043] Figure 3 Several structures of UDP sugar derivatives of galactosamine are shown, which can be modified at the 2-position with, for example, a 3-mercaptopropionyl group (2a), an azidoacetyl group (2b) or an azidodifluoroacetyl group (2c), or at the 6-position of N-acetylgalactosamine (2d) with an azido group.

[0044] Figure 4 Cyclooctyne (AT) is shown to be suitable for metal-free click chemistry and is the first choice for the reactive moiety Q.

[0045] Figure 5A set of exemplary toxic payloads for conjugation to various Trop-2-targeting monoclonal antibodies according to the present invention is shown. The point of attachment of the linker (to the amino group present in the payload) is indicated by an arrow. Preferred conjugates according to the present invention contain a payload, including a point of attachment, such as Figure 5 shown.

[0046] Figure 6 Shown are the structures of various camptothecins, which are preferred payloads in the context of the present invention.

[0047] Figure 7 Depicted are the structures of BCN linker drugs containing either Val-Cit-PABC or Val-Ala-PABC cleavable linkers for the preparation of ADCs via click chemistry of azidosugar remodeled antibodies with the following payloads (3 = ixotecan, 4 = MMAE, 5a = calicheamicin gamma 1 I , 5b = glycine calicheamicin gamma 1 I , 6 = 6-aminohexanoyl-maytansinoid). Linker-drug construct 3 is particularly preferred.

[0048] Figure 8 shows in vivo data of certolizumab-3 (DAR4) relative to DS-1062 in the N87 mouse xenograft model, a Phase 3 clinical ADC (DAR4) consisting of dedaburumab / hTINA antibody and derutecan linker payload (based on GGFG cleavable linker and DXd payload). After a single dose of 10 mg / kg (per dose), 18 days (Figure 8 (A)) or 31 days (Figure 8 (C)), it was noted that tumor regression of certolizumab-3 was significantly superior to dedaburumab-derutecan. Figure 8(B) and 8(D) Body weight graphs up to 18 or 31 days are shown.

[0049] Figure 9 shows the in vivo efficacy data of the Colo-205 mouse xenograft model. Both dose levels of hTINA-Drutecan showed low anti-tumor efficacy, while hRS7-3 showed significant anti-tumor efficacy. A clear dose response was observed, and the 8 mg / kg dose level even induced complete tumor regression from day 25 to the end of the study (day 70). Fig. 9B The Kaplan-Meier graph shows the changes in the body weight of mice over time. Fig. 9C shown.

[0050] FIG. 10 shows the HIC-HPLC trace of Example 12.

[0051] The present invention

[0052] In a first aspect, the present invention relates to an antibody conjugate having the general structure (1),

[0053] AB–[(L 6 ) b –{Z–L–D} x ] y (1)

[0055] in:

[0056] - AB is an antibody capable of targeting tumors expressing Trop-2;

[0057] -L is the linker connecting Z to D;

[0058] -Z is a linking group;

[0059] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w '-, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0060] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitin, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, indoline benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0061] -b is 0 or 1;

[0062] -x is 1 or 2; and

[0063] -y is 1, 2, 3, or 4.

[0064] The present invention also encompasses salts of the antibody conjugate according to structure (1), preferably pharmaceutically acceptable salts.

[0065] In a second aspect, the present invention relates to a method for preparing an antibody conjugate according to the present invention, the method comprising reacting a compound according to the general structure (2) with an antibody (3). The compound according to the general structure (2) comprises a reactive portion Q and an antibody reactive portion F, which antibody reactive portion F is capable of reacting with Q in a conjugation reaction, wherein Q and F react to form a linker Z. In this reaction, a conjugate according to the general structure (1) is formed. The method according to this aspect involves the following bioconjugation reaction:

[0066]

[0067] Below, the antibody conjugate according to structure (1) is first defined. The structural features of the antibody conjugate according to structure (1) are also applicable to the compound according to structure (2) and the antibody according to structure (3), because except for the reactive moieties F and Q, those are not changed in the conjugation reaction, which are converted to the linking group Z when the compound according to structure (2) reacts with the antibody according to structure (3).

[0068] In a third aspect, the present invention relates to the use of an antibody conjugate according to structure (1) for targeting cells expressing Trop-2. In relation thereto, the present invention relates to a first medical use and a second medical use of an antibody conjugate according to structure (1).

[0069] As will be appreciated by the skilled person, the definitions of chemical moieties and their preferred embodiments apply to all aspects of the invention.

[0070] General structure of antibody conjugates (1)

[0071] In a first aspect, the present invention relates to an antibody conjugate having the general structure (1):

[0072] AB–[(L 6 ) b –{Z–L–D} x ] y (1)

[0074] in:

[0075] - AB is an antibody capable of targeting tumors expressing Trop-2;

[0076] -b is 0 or 1;

[0077] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’-, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0078] -Z is a linking group;

[0079] -L is the linker connecting Z to D;

[0080] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitin, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, indoline benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0081] -x is 1 or 2; and

[0082] -y is 1, 2, 3, or 4.

[0083] Antibody AB

[0084] The antibody conjugate according to the present invention contains an antibody capable of targeting cells expressing Trop-2, in particular tumor cells. Trop-2 is a known target for cancer therapy. The term "expression" is used as commonly used in the art and refers to overexpression of a target relative to expression in healthy tissues. Antibodies capable of targeting tumors expressing Trop-2 may also be referred to as anti-Trop-2 antibodies, Trop-2 targeting antibodies or Trop-2 binding antibodies. Anti-Trop-2 antibodies selectively bind to cells expressing Trop-2.

[0085] Anti-Trop-2 antibodies are known in the art and any suitable antibody may be used in the context of the present invention. Preferred antibodies are selected from the list consisting of Huk5-70-2, hRS7, hTINA, AR47A6.4.2, RN926. Most preferably, the antibody is hRS7.

[0086] Here, the Fc region of these antibodies may have one or more mutations, such as 0 to 10 mutations or 0 to 5 mutations. Particularly preferred are mutations that alter binding to the FcRn receptor to modulate the half-life of the antibody. For example, the inclusion of mutations Met252Tyr, Ser254Thr, and Thr256Glu (commonly referred to as YTE) in IgG1Fc results in about 11-fold higher binding of the antibody to human FcRn, thereby extending the circulating half-life by about 3.5-fold. Thus, in one embodiment, the antibody has a half-life of less than 2.5 x 10 -6 M, preferably 0.05 to 0.99 x 10 -6 M, more preferably in the range of 0.1 to 0.49 x 10 -6 M, most preferably in the range of 0.2 to 0.4 x 10 -6 The apparent human FcRn binding affinity K in the range of D，表观 . Apparent binding affinity K D，表观 The assay can be performed according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, antibody AB is a YTE mutant of a preferred antibody as defined above or below.

[0087] hRS7 is also known as certolizumab, and can also be defined as containing a light chain sequence according to SEQ ID No.3 and a heavy chain sequence according to SEQ ID No.4, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably with exotecan as payload D.

[0088] hTINA is also known as dedabotulinumab and can also be defined as containing a light chain sequence according to SEQ ID No.7 and a heavy chain sequence according to SEQ ID No.8, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, most preferably with exotecan as payload D.

[0089] Alternatively, the antibody is L and V HThe variable domains are defined as domains that together form the variable domain that binds the antigen. Thus, in a preferred embodiment, the antibody comprises a V domain selected from the group consisting of SEQ ID No. 1, 5, 9, 13, 19, 21 and 23. L A domain and a V selected from the group consisting of SEQ ID No. 2, 6, 10, 14, 15, 20, 22 and 24 H In a further preferred embodiment, the antibody comprises a V domain of SEQ ID No. 1 or 5, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. L domain and V of SEQ ID No. 2 or 6 H domain, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a particularly preferred embodiment, the antibody comprises a V domain of SEQ ID No. 1. L domain and V of SEQ ID No.2 H Domains wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.

[0090] The above sequence identity refers to V L and V H The complete sequence of the structural domains. Although the entire sequence of these structural domains allows for some variation in sequence without impairing binding to Trop-2, it is preferred that the complementary determining regions (CDRs) have a high sequence identity to ensure that binding to Trop-2 is not significantly impaired. The positions of the CDRs are given in the table below. Therefore, preferably the antibody contains a V selected from the group consisting of SEQ ID No. 1, 5, 9, 13, 19, 21 and 23. L A domain, and a V selected from the group consisting of SEQ ID No. 2, 6, 10, 14, 15, 20, 22 and 24. H domain, preferably V of SEQ ID No. 1 or 5 L domain, and V of SEQ ID No. 2 or 6 H domain, more preferably V of SEQ ID No.1 L domain, and V of SEQ ID No.2 H The sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. It is understood by those skilled in the art that the VL and V H The domains can be combined with appropriate constant domains to form complete antibodies.

[0091] Preferred V L Domain

[0092] SEQ ID No. CDR (amino acid number within the sequence) 1 24-34|50-56|89-97 5 24-34|50-56|89-97 9 24-34|50-56|89-97 13 24-34|50-56|89-97 19 24-34|50-56|89-97 21 24-34|50-56|89-97 23 24-34|50-56|89-97

[0093] Preferred V H Domain

[0094] SEQ ID No. CDR (amino acid number within the sequence) 2 31-35|50-66|99-110 6 31-35|50-66|99-110 10 31-35|50-66|99-110 14 31-35|50-66|99-110 15 31-35|50-66|99-110 20 31-35|50-66|99-110 22 31-35|50-66|99-110 24 31-35|50-66|99-110

[0095] In a particularly preferred embodiment, the antibody comprises V of SEQ ID No. 1 L domain and V of SEQ ID No.2 H domain, or V of SEQ ID No.5 L domain and V of SEQ ID No.6 H domain, or V of SEQ ID No.9 L domain and V of SEQ ID No.10 H domain, or V of SEQ ID No.13 L domain and V of SEQ ID No. 14 or 15 H domain, or V of SEQ ID No.19 L domain and V of SEQ ID No.20 H domain, or V of SEQ ID No.21 L domain and V of SEQ ID No.22 H domain, or V of SEQ ID No.23 L domain and V of SEQ ID No.24 H Herein, the sequence identity defined above for the complete sequence as well as for the CDRs applies. Most preferably, the antibody comprises V of SEQ ID No. 1 L domain and V of SEQ ID No.2 H Structural domain.

[0096] Alternatively, the antibody is defined by its light chain and heavy chain, which together form the antibody. Thus, in a preferred embodiment, the antibody contains a light chain selected from the group consisting of SEQ ID No. 3, 7, 11 and 16, and a heavy chain selected from the group consisting of SEQ ID No. 4, 8, 12, 17 and 18, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a more preferred embodiment, the antibody contains a light chain of SEQ ID No. 3 or 7, and a heavy chain selected from the group consisting of SEQ ID No. 4 or 8, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a most preferred embodiment, the antibody contains a light chain of SEQ ID No. 3, and a heavy chain selected from the group consisting of SEQ ID No. 4, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.

[0097] The above sequence identities refer to the complete sequence of the light chain and the heavy chain. Although the entire sequence of these chains allows for some variation in the sequence without impairing binding to Trop-2, it is preferred that the CDRs have a higher sequence identity to ensure that binding to Trop-2 is not significantly impaired. The following table gives the positions of the CDRs. Therefore, it is preferred that the antibody contains a light chain selected from the group consisting of SEQ ID No. 3, 7, 11 and 16, and a heavy chain selected from the group consisting of SEQ ID No. 4, 8, 12, 17 and 18, wherein the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

[0098] Preferred light chain

[0099] SEQ ID No. CDR (amino acid number within the sequence) 3 24-34|50-56|89-97 7 24-34|50-56|89-97 11 24-34|50-56|89-97 16 24-34|50-56|89-97

[0100] Preferred heavy chain

[0101] SEQ ID No. CDR (amino acid number within the sequence) 4 31-35|50-66|99-110 8 31-35|50-66|99-110 12 31-35|50-66|99-110 17 31-35|50-66|99-110 18 31-35|50-66|99-110

[0102] In a particularly preferred embodiment, the antibody contains a light chain of SEQ ID No.3 and a heavy chain of SEQ ID No.4, or a light chain of SEQ ID No.7 and a heavy chain of SEQ ID No.8, or a light chain of SEQ ID No.11 and a heavy chain of SEQ ID No.12, or a light chain of SEQ ID No.16 and a heavy chain of SEQ ID No.17 or 18. Herein, the sequence identities defined above for the complete sequence and for the CDRs apply. Most preferably, the antibody comprises a light chain of SEQ ID No.3 and a heavy chain of SEQ ID No.4, wherein the sequence identities defined above for the complete sequence and for the CDRs apply. Particularly preferred is such an antibody with exitecan as payload D.

[0103] Connector L 6

[0104] If the reactive group F is directly attached to the antibody, or even part of the antibody structure, there is no linker L connecting AB to F (for the antibody of structure (3)) or connecting AB to Z (for the conjugate of structure (1)). 6 and b = 0. This is the case, for example, for cysteine ​​conjugation and lysine conjugation. Alternatively, a linker L may be used 6 A reactive group F is introduced onto the antibody, and a linker connects AB to F (for the antibody of structure (3)) or to Z (for the conjugate of structure (1)), in which case L is present. 6 And b = 1. If there is L 6 , then a reactive group F is typically introduced at the glycan of the antibody. This is the case, for example, of conjugation via artificially introduced reactive groups F, such as using transglutaminase or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyl-transferase). For example, a modified sugar residue S(F) may be introduced at the glycan x , extending the glycan with one monosaccharide residue S, which introduces x reactive groups F on the glycan of the antibody. In the most preferred embodiment, the conjugation occurs via the glycan of the antibody and b = 1. The conjugation site is preferably located at the heavy chain of the antibody.

[0105] If present, L 6 It is the linker that connects AB to F or Z and consists of –GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’- represents, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–. Usually, L 6 At least in part, it is formed by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at or near the asparagine residue at position 297 of the heavy chain, which is modified by a complex type of glycan. It is preferred to use the naturally occurring glycosylation site of the antibody, but other glycosylation sites, including artificially introduced glycosylation sites, can also be used in the linker L. 6 Therefore, in a preferred embodiment, L 6 An amino acid located within the range of 250 to 350 of the heavy chain, preferably within the range of 280 to 310 of the heavy chain, more preferably within the range of 295 to 300 of the heavy chain, most preferably at position 297 of the heavy chain is linked to the antibody.

[0106] L 6 -GlcNAc(Fuc) w –(G) j – is a glycan or part thereof of an antibody. Therefore, the –GlcNAc(Fuc) w –(G) j – Typically derived from the original antibody, where GlcNAc is the N-acetylglucosamine moiety and Fuc is the fucose moiety. Fuc is typically bound to GlcNAc via an α-1,6-glycosidic bond. Typically, an antibody may (w=1) or may not (w=0) be fucosylated. In the context of the present invention, the presence of the fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. GlcNAc residues may also be referred to as core-GlcNAc residues, and are monosaccharides that are directly attached to the peptide portion of the antibody.

[0107] S can be directly linked to the core-GlcNAc(Fuc) w part, i.e. j = 0, which means that before S attachment, the remaining part of the glycan is separated from the core-GlcNAc(Fuc) w This type of polysaccharide trimming is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, in the core-GlcNAc(Fuc) w There are one or more monosaccharide residues between moiety and S, i.e. j is an integer in the range of 1 to 10, preferably j=2 to 5. In a preferred embodiment, (G) jis an oligosaccharide moiety comprising j monosaccharide residues G, wherein j is an integer ranging from 2 to 5. (G) j Usually linked to GlcNAc(Fuc) via a β-1,4 bond w GlcNAc part. In a preferred embodiment, j is 3, 4 or 5. Although any monosaccharide that may be present in the polysaccharide can be used as G, each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose and N-acetylneuraminic acid. More preferably, G is selected from galactose, N-acetylglucosamine, mannose. The inventors have found that antibody conjugates with j below 4 do not show or hardly any binding to Fc-γ receptors, while antibody conjugates with j in the range of 4 to 10 do bind to Fc-γ receptors. Therefore, by selecting a certain value of j, the desired degree of binding to Fc-γ receptors can be obtained. Therefore, preferably j = 0, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably j = 0, 3, 4 or 5, most preferably the antibody is trimmed and j = 0.

[0108] S is sugar or sugar derivative.Term " sugar derivative " is used to represent the derivative of monosaccharide in this article, i.e. the monosaccharide containing substituent and / or functional group.Suitable example of S comprises glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugar and sugar acid, such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (it is also called N-acetylneuraminic acid (NeuNAc) and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA).Preferably, S is selected from Glc, Gal, GlcNAc and GalNAc.In a particularly preferred embodiment, S is GalNAc.

[0109] x is an integer representing the number of linking groups Z (for conjugate (1)) or reactive groups F (for antibody (3)) linked to the sugar (derivative) S. Thus, the antibody according to the invention contains a moiety S comprising x reactive moieties F. Each of these reactive moieties F reacts with a reactive moiety Q of a compound according to general structure (2) such that x linking groups Z are formed and x compounds according to general structure (2) are linked to a single occurrence of S. x is 1 or 2, preferably x=1.

[0110] The linking group Z (for the conjugate (1)) or the reactive group F (for the antibody (3)) may be directly linked to S, or a linker L may be present between S and Z or F. 7 .L 7It is the linker connecting S and Z. It can be present (w'=1 or 2) or absent (w'=0) L 7 Typically, each part Z can be connected via a linker L 7 Connected to S, so in one embodiment x has w'=0. Preferably, there is no L 7 And each connecting part Z is directly connected to S. If present, L 7 It can be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. In a preferred embodiment, x=1 and w'=0 or 1, most preferably x=1 and w'=0.

[0111] y is an integer representing the number of sugar (derivative) S, each sugar (derivative) S having x reactive groups F or connected to x linking groups Z, which are connected to the antibody. y is 1, 2, 3 or 4, preferably y=2 or 4, most preferably y=2. Therefore, the antibody contains y parts S, each of which contains x reactive parts F. Each of these reactive parts F reacts with the reactive part Q of the compound according to the general structure (2), so that x+y linking groups Z are formed, and x+y compounds according to the general structure (2) are connected to a single antibody. For example, due to the branched nitrogen atom N* in L, each compound according to the general structure (2) can contain multiple payloads. It is preferred that each compound according to the general structure (2) contains 1 or 2 occurrences of D, most preferably 2 occurrences of D. In a particularly preferred embodiment, the linker L 1 Contains a second occurrence of a D-attached branch nitrogen atom N*.

[0112] The amount of payload (D) molecules attached to a single antibody is referred to in the art as DAR (drug-antibody ratio). In the context of the present invention, preferably DAR is an integer in the range of 1 to 8, more preferably 2 or 4, most preferably DAR=4. In other words, DAR is preferably an integer in the range of (x+y) to [(x+y)×2], most preferably DAR=[(x+y)×2]. When the preferred value of x is 1 and the preferred value of y is 2, DAR is preferably 4. It should be understood that these are theoretical DAR values, and in practice, DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as random mixtures of antibody-drug conjugates, with different DAR values ​​between each conjugate, and DAR may have a wide distribution (e.g., DAR=0 to 10) or a narrow distribution (e.g., DAR=3 to 4) depending on the conjugation technology used. In the case of such mixtures, DAR generally refers to the average DAR of the mixture. This is well known in the field of bioconjugation. However, in cases where conjugation occurs via the glycan (ie b = 1 and L is present 6), the antibody conjugate according to the present invention has a DAR close to the theory. For example, when the theoretical DAR is 4, it is easy to obtain a DAR value higher than 3.6 or even higher than 3.8, which indicates that most of the antibodies in the reaction mixture have been completely reacted and the DAR is 4.

[0113] Linking group Z

[0114] Z is a linking group that covalently links the two parts of the conjugate according to the present invention. The term "linking group" herein refers to a structural element that is produced by the reaction between Q and F and that connects one part of the conjugate to another part of the same conjugate. As will be appreciated by those skilled in the art, the nature of the linking group depends on the type of reaction that obtains the connection between the parts of the compound. For example, when the carboxyl group of RC(O)-OH reacts with the amino group of H2N-R' to form RC(O)-N(H)-R', R is connected to R' via the linking group Z and Z can be represented by the group -C(O)-N(H)-. Since the linking group Z is derived from the reaction between Q and F, it can take any form.

[0115] Since more than one reactive part F can be present or introduced into the antibody, the antibody conjugate according to the present invention can contain more than one payload D in each biomolecule, for example 1 to 8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D. Taking into account the symmetrical nature of antibodies, the number of payloads is usually an even number. In other words, when one side of the antibody is functionalized by F, the symmetrical counterpart will also be functionalized. Alternatively, if the naturally occurring thiol group of the cysteine ​​residue of the protein is used as F, the value of m can be any value and can vary between individual conjugates.

[0116] In the compound according to structure (1), the linker Z is optionally connected via a linker L. 6 D is connected to AB. Many reactions for connecting reactive groups Q to reactive groups F are known in the art. Therefore, a variety of linking groups Z may be present in the conjugate according to the present invention. In one embodiment, the reactive group Q is selected from the above options, preferably as Figure 1 As shown, the complementary reactive groups F and the linking groups Z obtained therefrom are known to those skilled in the art. Figure 1 It is shown that when a linker-conjugate comprising Q is conjugated to a biomolecule comprising a complementary reactive group F, F and Q as well as the linking group Z that will be present in the bioconjugate 3 Several examples of suitable combinations of .

[0117] For example, when F comprises or is a thiol group, the complementary group Q comprises an N-maleimido group and an alkenyl group, and the corresponding linking group Z is as follows Figure 1 When F comprises or is a thiol group, the complementary group Q further comprises an amide group.

[0118] For example, when F comprises or is an amino group, the complementary group Q includes a keto group and an activated ester group, and the corresponding linking group Z is such as Figure 1 shown.

[0119] For example, when F comprises or is a keto group, the complementary group Q includes an (O-alkyl)hydroxyamino group and a hydrazine group, and the corresponding linking group Z is such as Figure 1 shown.

[0120] For example, when F comprises or is an alkynyl group, the complementary group Q comprises an azido group, and the corresponding linking group Z is such as Figure 1 shown.

[0121] For example, when F comprises or is an azido group, the complementary group Q comprises an alkynyl group, and the corresponding linking group Z is such as Figure 1 shown.

[0122] For example, when F comprises or is a cyclopropenyl group, a trans-cyclooctenyl group or a cyclooctyne group, the complementary group Q comprises a tetrazinyl group, and the corresponding linking group Z is such as Figure 1 In these specific cases, Z is only an intermediate structure and N2 will be eliminated to produce a dihydropyridazine (from the reaction with the olefin) or a pyridazine (from the reaction with the alkyne).

[0123] Other suitable combinations of F and Q and the properties of the resulting linking group Z3 are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), in particular Chapter 3, pages 229-258, which are incorporated by reference. A list of complementary reactive groups suitable for bioconjugation methods is disclosed in Table 3.1, Chapter 3, pages 230-232 of GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), and the contents of the table are expressly incorporated herein by reference.

[0124] In a preferred embodiment, the linker group Z is obtained by a cycloaddition or nucleophilic reaction, preferably wherein the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition, or the nucleophilic reaction is a Michael addition or a nucleophilic substitution. This cycloaddition or nucleophilic reaction occurs via a reactive group F connected to S and a reactive group Q connected to D via L. Conjugation reactions via cycloaddition or nucleophilic reactions are known to those skilled in the art, and those skilled in the art are able to select appropriate reaction partners F and Q, and will understand the properties of the resulting linker group Z.

[0125] In a first preferred embodiment, Z is formed by a cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reactions) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are reverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and those skilled in the art know how to perform them.

[0126] Preferably, Z contains a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide or imide groups. The triazole moiety is particularly preferably present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e., is formed by Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e., is formed by Q comprising a (hetero)cycloalkene moiety. In a preferred embodiment, Z has the structure (Z1):

[0127]

[0128] Here, it is represented as The bonds are either single or double. In addition:

[0129] - Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za)-(Zj) defined below, wherein the carbon atom marked with ** corresponds to the carbon atom of (Z1) fused to ring Z represented by The bond between the two carbon atoms;

[0130] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0131] –C 24 The group consisting of (hetero)arylalkyl groups;

[0132] -Y 2 is C(R 31 )2. O. S. S (+) R 31 、S(O)R 31 、S(O)=NR 31 or NR 31 , where S (+) It was B (-) The balanced cationic sulfur atom, where B (-) is an anion, and each R 31 R alone 15 or a connection to D, which is connected via L;

[0133] -u is 0, 1, 2, 3, 4, or 5;

[0134] -u' is 0, 1, 2, 3, 4 or 5, wherein u+u'=0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0135] -v=an integer in the range of 8 to 16;

[0136] - Ring A is formed by cycloaddition and is preferably selected from (Za)-(Zj).

[0137] If depicted as If the bond of is a double bond, then preferably u+u'=4, 5, 6, 7 or 8. Preferably, the wavy bond marked with * is connected to S, and the wavy bond marked with ** is connected to L.

[0138] Particularly preferably, Z comprises a (hetero)cycloalkene moiety, i.e. depicted as The bond is a double bond. In a preferred embodiment, Z is selected from the structures (Z2)-(Z20), as shown below:

[0139]

[0140] Here, the connection to L is depicted by a wavy bond. (–) It is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za)-(Zm) as depicted below, wherein the carbon atoms marked with ** correspond to two carbon atoms of the (hetero)cycloalkane ring (Z2)-(Z20) to which ring Z is fused. Preferably, ring Z is selected from (Za)-(Zj), more preferably selected from (Za), (Zd) and (Zh), and most preferably, ring Z has structure (Za). Since in the context of the present embodiment, the linking group Z is formed by reaction with a (hetero)cycloalkyne, the above depiction is The bond is a double bond.

[0141]

[0142] In further preferred embodiments, Z is selected from structures (Z21)-(Z38) and (Z8a), as shown below:

[0143]

[0144]

[0145] Here, the connection to L is depicted by a wavy bond. In structure (Z38), B (–) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za)-(Zm) as defined above, preferably from structures (Za)-(Zj).

[0146] In preferred embodiments, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to Structure (Z8), (Z26), (Z27), (Z28) or (Z37) or (Z38a), more preferably according to Structure (Z8), (Z26), (Z27), (Z28) or (Z37), which is optionally substituted. Each of these preferred options for Z is further defined below.

[0147] Thus, in preferred embodiments, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Z37) is unsubstituted.

[0148] In preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z is structure (Z42) as further defined below.

[0149] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27) or (Z28), which is optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 are independently selected from hydrogen, a linear or branched C1-C 12 Alkyl group or C4-C 12 The aromatic ring in (Z40) is optionally O-sulfonylated at one or more positions, while the ring of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctenyl moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is structure (Z43) as further defined below.

[0150] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37).

[0151]

[0152] In particularly preferred embodiments, Z comprises a cyclooctenyl group and is according to structure (Z42):

[0153]

[0154] In this article:

[0155] - The bond marked with * is connected to S, and the wavy bond marked with ** is connected to L;

[0156] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alk(hetero)aryl and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–

[0157] C 24 The group consisting of (hetero)arylalkyl groups;

[0158] -R 18 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups;

[0159] -R 19 Selected from hydrogen, halogen, C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, the alkyl groups are optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Z (or Q) or D attached via a spacer moiety; and

[0160] -l is an integer in the range 0 to 10.

[0161] In preferred embodiments of the group according to structure (Z42), R 15 are independently selected from hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In preferred embodiments of the group according to structure (Z42), R 18 R is independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably two R 18 are all H. In preferred embodiments of the group according to structure (Z42), R 19 is H. In preferred embodiments of the group according to structure (Z42), l is 0 or 1, more preferably l is 1.

[0162] In particularly preferred embodiments, Z comprises a (hetero)cyclooctynyl group and is according to structure (Z43):

[0163]

[0164] In this article:

[0165] - The bond marked with * is connected to S, and the wavy bond marked with ** is connected to L;

[0166] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0167] –C 24 The group consisting of (hetero)arylalkyl groups;

[0168] -Y is N or CR 15 .

[0169] In preferred embodiments of the group according to structure (Z43), R 15are independently selected from hydrogen, halogen, -OR 16 、-S(O)3 (-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from hydrogen and -S(O)3 (-) In a preferred embodiment of the group according to structure (Z43), Y is N or CH, more preferably Y=N.

[0170] In particularly preferred embodiments, Z comprises a heterocycloheptyne group and is according to structure (Z37) or (Z38a), preferably according to structure (Z37), wherein ring Z is a triazole ring:

[0171]

[0172] In an alternative preferred embodiment, Z comprises a (hetero)cycloalkane moiety, i.e., depicted as The key is a single bond. (Hetero) cycloalkyl group may also be referred to as heterocycloalkyl group or cycloalkyl group, preferably cycloalkyl group, wherein the (hetero) cycloalkyl group is optionally substituted. Preferably, the (hetero) cycloalkyl group is a (hetero) cyclopropyl group, a (hetero) cyclobutyl group, a norbornyl group, a norbornyl group, a (hetero) cycloheptyl group, a (hetero) cyclooctyl group, a (hetero) cyclononyl group or a (hetero) cyclodecyl group, all of which may be optionally substituted. Particularly preferred are (hetero) cyclopropyl group, (hetero) cycloheptyl group or (hetero) cyclooctyl group, wherein the (hetero) cyclopropyl group, trans-(hetero) cycloheptyl group or (hetero) cyclooctyl group are optionally substituted. Preferably, Z comprises a cyclopropyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornene or norbornene group according to structure (Z46), a (hetero)cycloheptyl moiety according to structure (Z47) or a (hetero)cyclooctyl moiety according to structure (Z48). Here, Y 3 Selected from C(R 23 )2. NR 23 or O, where each R 23 Each is hydrogen, C1-C6 alkyl or optionally connected to L via a spacer, and is marked as The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropyl group is according to structure (Z49). In another preferred embodiment, the (hetero)cycloheptyl group is according to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group is according to structure (Z52), (Z53), (Z54), (Z55) or (Z56).

[0173]

[0174] Here, the R group on Si in (Z50) and (Z51) is typically an alkyl or aryl group, preferably a C1-C6 alkyl group. Ring Z is typically selected from the structures (Zn)-(Zu), wherein the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z44)-(Z56) fused to ring Z, and the carbon marked with * is directly connected to the peptide chain of the antibody. Preferably, ring Z is selected from (Zo)-(Zr). Since the linker Z in the context of this embodiment is formed by reaction with a (hetero)cycloalkene, it is depicted above as The bond is a single bond.

[0175]

[0176]

[0177] In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably by nucleophilic substitution or Michael addition, preferably by Michael addition. The preferred Michael reaction is a sulfhydryl-maleimide connection, most preferably wherein Q is maleimide and F is a sulfhydryl group. Preferably, the sulfhydryl group is present in the side chain of a cysteine ​​residue. In a preferred embodiment, the linking group Z comprises a succinimidyl ring or an open-ring succinamide derivative thereof. Preferred options for the linking group Z include a portion selected from (Z57)-(Z71) depicted below.

[0178]

[0179] Herein, the wavy bonds marked with * are optionally connected to the antibody Ab via a linker, and the wavy bonds without a label are optionally connected to the payload via a linker. 29 It is C 1-12 Alkyl, preferably C 1-4 alkyl, most preferably ethyl, and X 1 is O or S, preferably X 1 =0. The nitrogen atoms marked with ** in (Z67)-(Z71) correspond to the nitrogen atoms of the side chains of the antibody lysine residues. The carbon atoms of the phenyl groups of (Z69) and (Z70) are optionally substituted, preferably optionally fluorinated.

[0180] In a preferred embodiment, the linker group Z comprises a moiety selected from (Z1)-(Z71).

[0181] Connector L

[0182] The linker L connects the payload D to the linking group Z (in the conjugate according to structure (1)) or connects the payload D to the reactive group Q (in the compound according to structure (2)). Linkers are known in the art and may be cleavable or non-cleavable. The linker L preferably contains a self-immolative group or a cleavable linker comprising a peptide spacer and a p-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0183] In a preferred embodiment, the linker L is of the structure –(L 1 ) n –(L 2 ) o –(L 3 ) p –(L 4 ) q –, where (L 4 ) q connected to the payload D and (L 1 ) n Connect to Z or Q. In this article, L 1 , L 2 , L 3 and L 4 is a linker or connecting unit and each of n, o, p and q is independently 0 or 1, wherein n+o+p+q is at least 1. In a preferred embodiment, at least one linker L is present. 1 and L 2 (ie n = 1; o = 1; p = 0 or 1; q = 0 or 1), preferably there is a linker L 1 , L 2 and L 3 and the presence or absence of L 4 (ie n=1; o=1; p=1; q=0 or 1). In one embodiment, there is a linker L 1 , L 2 , L 3 and L 4 (ie n=1; o=1; p=1; q=1). In one embodiment, there is a linker L 1 , L 2 and L 3 And there is no L 4 (ie n=1; o=1; p=1; q=0).

[0184] Connectors, especially connector L 1, may contain one or more branch points for connecting multiple payloads to a single linker. In a preferred embodiment, the linker of the conjugate according to the present invention contains a branching portion. A "branching portion" in the context of the present invention refers to a portion embedded in the linker connecting three parts. In other words, the branching portion contains at least three bonds to other parts, usually one bond to Z or Q, one bond to payload D and one bond to a second payload D. If a branching portion is present, it is preferably embedded in the linker L. 1 More preferably, Sp 2 Part of or as NR 13 Any moiety containing at least three bonds to other moieties is suitable as a branching moiety in the context of the present invention. In a preferred embodiment, the branching moiety is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring or a polycyclic moiety. Most preferably, the branching moiety is a nitrogen atom.

[0185] Connector L 1

[0186] The absence (n=0) or presence (n=1) of linker L 1 Preferably, there is a linker L 1 And n = 1. 1 For example, it can be selected from a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkyne group, C3-C 200 Cycloalkylene groups, C5-C 200 Cycloalkenylene groups, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 Optionally, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally, the groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, preferably selected from O, S(O) y’ and NR 21 The group consisting of, wherein y' is 0, 1 or 2, preferably y'=2, and R 21 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups.

[0187] In a preferred embodiment, the connector L 1 Contains polar groups. Such polar groups can be selected from (poly)ethylene glycol diamines (e.g. 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains and 1,z'-diaminoalkanes (wherein z' is the number of carbon atoms in the alkane, preferably z'=1-10), -(O) a –C(O)–NH–S(O)2–NR 13 – (as further defined below, see structure (23)), –C(S(O)3 (–) )–,–C(C(O)2 (–) )–、–S(O)2–、–P(O)2 (–) –, –O(CH2CH2O) t –、–NR 30 (CH2CH2NR 30 ) t – and the following two structures:

[0188]

[0189] The polar group may also contain an amino acid preferably selected from Arg, Glu, Asp, Ser and Thr. Here, for structure (23), a and R 13 Further defined as follows. t is an integer in the range of 0 to 15, preferably 1 to 10, more preferably 2 to 5, most preferably t=2 or 4. Each R 30 Independently H, C 1-12 Alkyl, C 1-12 Aryl, C 1-12 Alkyl or C 1-12 Arylalkyl. Linker L 1 There may be more than one such polar group, for example at least two polar groups. Polar groups may also be present in the linker L. 1 In the branches of, which branch from the branching moiety as defined elsewhere. Preferably, nitrogen or carbon atoms are used as branching moieties. Particularly preferably, -O(CH2CH2O) is present in the branches t –Polar groups.

[0190] In a preferred embodiment, the connector L 1 is or comprises a sulfonamide group, preferably a sulfonamide group according to structure (23):

[0191]

[0192] The wavy line represents the connection to the rest of the compound, usually to Q and L. 2 , L 3 , L 4 or D, preferably with Q and L 2 Preferably, (O) a The C(O) part is connected to Q and NR 13 Partially connected to L 2 , L 3 , L 4 or D, preferably connected to L 2 .

[0193] In structure (23), a=0 or 1, preferably a=1, and R 13 Selected from hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The group consisting of (hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The (hetero)arylalkyl group is optionally substituted and optionally replaced by one or more selected from O, S and NR 14 The heteroatom interval, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is via a spacer moiety (preferably Sp as defined below) 2 ) is connected to N. In one embodiment, D is connected to N via – (B) e –(A) f –(B) g – C(O)-connected to N.

[0194] In a preferred embodiment, R 13 is hydrogen or C1-C 20 An alkyl group, more preferably R 13 is hydrogen or C1-C 16 An alkyl group, even more preferably R 13 is hydrogen or C1-C 10 An alkyl group, wherein the alkyl group is optionally substituted and optionally substituted by one or more selected from O, S and NR 14of heteroatoms, preferably O, wherein R 14 In a preferred embodiment, R 13 In another preferred embodiment, R 13 It is C1-C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 An alkyl group, wherein the alkyl group is optionally interrupted by one or more O atoms, and wherein the alkyl group is optionally substituted by an -OH group, preferably a terminal -OH group. In this embodiment, further preferably R 13 is a (poly)ethylene glycol chain containing a terminal -OH group. In another preferred embodiment, R 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and isopropyl, and even more preferably selected from the group consisting of hydrogen, methyl and ethyl. Still even more preferably, R 13 is hydrogen or methyl, and most preferably R 13 It's hydrogen.

[0195] In a preferred embodiment, L 1 According to structure (24):

[0196]

[0197] In this paper, a and R 13 As defined above, Sp 1 and Sp 2 is independently a spacer moiety and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, more preferably b=0 and c=1. In one embodiment, the spacer Sp 1 and Sp 2 Independently selected from linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkyne group, C3-C 200 Cycloalkylene groups, C5-C 200 Cycloalkenylene groups, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylene groups and C9-C 200The group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group and an arylalkynylene group are optionally substituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatom interval, where R 16 are independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of alkyl, alkenyl, alkynyl and cycloalkyl groups is optionally substituted. When alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are interrupted by one or more heteroatoms as defined above, preferably the groups are interrupted by one or more O atoms and / or by one or more SS groups.

[0198] More preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkyne group, C3-C 100 Cycloalkylene groups, C5-C 100 Cycloalkenylene groups, C8-C 100 Cycloalkynylene group, C7-C 100 Alkyl arylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylene groups and C9-C 100 The group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group and an arylalkynylene group are optionally substituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatom interval, where R 16 are independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of a cycloalkyl group, said alkyl group, alkenyl group, alkynyl group and cycloalkyl group are optionally substituted.

[0199] Even more preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkyne group, C3-C 50 Cycloalkylene groups, C5-C 50 Cycloalkenylene groups, C8-C 50 Cycloalkynylene group, C7-C 50 Alkyl arylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylene groups and C9-C 50 The group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group and an arylalkynylene group are optionally substituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatom interval, where R 16 are independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of a cycloalkyl group, said alkyl group, alkenyl group, alkynyl group and cycloalkyl group are optionally substituted.

[0200] Still even more preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkyne group, C3-C 20 Cycloalkylene groups, C5-C 20 Cycloalkenylene groups, C8-C 20 Cycloalkynylene group, C7-C 20 Alkyl arylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylene groups and C9-C 20The group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group and an arylalkynylene group are optionally substituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatom interval, where R 16 are independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of a cycloalkyl group, said alkyl group, alkenyl group, alkynyl group and cycloalkyl group are optionally substituted.

[0201] In these preferred embodiments, it is further preferred that the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group are unsubstituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatom-interval consisting of a group of 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0202] Most preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 20 The group consisting of an alkylene group, the alkylene group is optionally substituted and optionally one or more selected from O, S and NR 16 The heteroatom-interval consisting of a group of 16 are independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of alkyl, alkenyl, alkynyl and cycloalkyl groups, wherein the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. In this embodiment, it is further preferred that the alkylene group is unsubstituted and is optionally substituted with one or more selected from O, S and NR 16 The heteroatoms (preferably O and / or SS) are spaced apart, wherein R 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0203] Preferred spacer moieties Sp 1 and Sp 2 So including -(CH2) r-、-(CH2CH2) r -、-(CH2CH2O) r -、-(OCH2CH2) r -、-(CH2CH2O) r CH2CH2-、-CH2CH2(OCH2CH2) r -、-(CH2CH2CH2O) r -、-(OCH2CH2CH2) r -、-(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r -, wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20 and yet even more preferably in the range of 1 to 15. More preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, and even more preferably 1, 2, 3 or 4.

[0204] Alternatively, the preferred linker L 1 Can be - (W) k –(A) d –(B) e –(A) f –(C(O)) g – means, where:

[0205] - d = 0 or 1, preferably d = 1;

[0206] - e = an integer in the range of 0 to 10, preferably e = 0, 1, 2, 3, 4, 5 or 6, preferably an integer in the range of 1 to 10, most preferably e = 1, 2, 3 or 4;

[0207] - f = 0 or 1, preferably f = 0;

[0208] - wherein d+e+f is at least 1, preferably in the range of 1 to 5; and preferably, wherein d+f is at least 1, preferably d+f=1.

[0209] - g = 0 or 1, preferably g = 1;

[0210] - k = 0 or 1, preferably k = 1;

[0211] -A is a sulfonamide group according to structure (23);

[0212] -B is a –CH2–CH2–O– or –O–CH2–CH2– moiety, or (B)e Yes – (CH2–CH2–O) e1 –CH2–CH2–

[0213] or –(CH2–CH2–O) e1 –CH2– moiety, wherein e1 is defined the same as e;

[0214] -W is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–,

[0215] –C(O)(CH2) m C(O)–, –C(O)(CH2) m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably wherein W is -OC(O)NH-, -C(O)(CH2) m C(O)NH— or —C(O)NH—, and wherein m is an integer in the range of 0 to 10, preferably m=0, 1, 2, 3, 4, 5 or 6, most preferably m=2 or 3;

[0216] - Preferably, wherein L 1 Via(W) k Connected to Q and via (C(O)) g , preferably connected to L via C(O) 2 , L 3 , L 4 or D, preferably connected to L 2 .

[0217] In the context of this example, the wavy line in structure (23) indicates that the adjacent groups, such as (W) k (B) e and (C(O)) g Preferably A is according to structure (23), wherein a=1 and R 13 =H or C1-C 20 An alkyl group, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0218] Preferred connector L 1 With structure – (W) k –(A) d –(B) e –(A) f –(C(O)) g -,in:

[0219] (a) k=0; d=1; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2.

[0220] (b) k = 1; W = –C(O)(CH2) m C(O)NH–;m=2;d=0;(B) e =–(CH2–CH2–O) e1 –CH2–CH2–; f=0; g=1; e1=1, 2, 3 or 4, preferably e=1.

[0221] (c) k=1; W=-OC(O)NH-; d=0; B=-CH2-CH2-O-; g=1; f=0; e=1, 2, 3 or 4, preferably e=2.

[0222] (d) k = 1; W = –C(O)(CH2) m C(O)NH–;m=2;d=0;(B) e =–(CH2–CH2–O) e1 –CH2–CH2–; f=0; g=1; e1=1, 2, 3 or 4, preferably e1=4.

[0223] (e)k=1; W=–OC(O)NH–; d=0; (B) e =–(CH2–CH2–O) e1 –CH2–CH2–; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4.

[0224] (f)k=1; W=–(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, m=3; d=0; (B) e =–(CH2–CH2–O) e1 –CH2–CH2–; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4.

[0225] (g) k=0; d=0; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2.

[0226] (h) k=1; W=—C(O)NH—; d=0; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2.

[0227] In a preferred embodiment, the connector L 1 Contains branched nitrogen atoms, which are located between Q or Z and (L 2 ) oand contains a further moiety D as a substituent, which is preferably connected to the branching nitrogen atom via a linker. An example of a branching nitrogen atom is the nitrogen atom NR in structure (23) 13 , where R 13 is connected to the second occurrence of D via a spacer moiety. Alternatively, the branching nitrogen atom may be located according to the structure - (W) k –(A) d –(B) e –(A) f –(C(O)) g –L 1 In one embodiment, L 1 By – (W) k –(A) d –(B) e –(A) f –(C(O)) g –N*[–(A) d –(B) e –(A) f –(C(O)) g’ –]2 represents, wherein A, B, W, d, e, f, g and k are as defined above and are selected individually at each occurrence, and N* is a branching nitrogen atom, –(A) d –(B) e –(A) f –(C(O)) g’ Both instances of – are connected to it. Here, both (C(O)) g’ Some are connected to –(L 2 ) o –(L 3 ) p –(L 4 ) q –D, where L 2 , L 3 , L 4 , o, p, q and D are as defined above, and each is selected independently. In a preferred embodiment, L 2 , L 3 , L 4 Each of o, p, q and D is equivalent to (C(O)) g’ The two parts of the connection are the same.

[0228] Preferred linkers L containing branched nitrogen atoms 1 With structure – (W) k –(A) d –(B) e –(A) f –(C(O)) g–N*[–(A') d’ –(B') e’ –(A') f’ –(C(O)) g’ –]2, where:

[0229] (i) k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is according to structure (23), where a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0230] (j) k = d = g = e' = g' = 1; f = d' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is according to structure (23), where a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0231] Connector L 2

[0232] The absence (o=0) or presence (o=1) of the linker L 2 Preferably, there is a linker L 2 And o = 1. Joint L 2 is a peptide spacer. The peptide spacer is preferably composed of (NH-CR 17 –CO) n Definition, where R 17Represents an amino acid side chain known in the art. Here, the amino acid can be a natural or synthetic amino acid. Preferably, the amino acids are all in L-configuration. n is an integer in the range of 1 to 5, preferably an integer in the range of 2 to 5. Therefore, the peptide spacer preferably contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), a tripeptide (n=3) or a tetrapeptide (n=4), and most preferably the peptide spacer is a dipeptide. Although any peptide spacer can be used, preferably the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-L In one embodiment, AcLys is acetyl-lysine, and iGlu is isoglutamic acid. In one embodiment, L 2 =Val-Cit. In one embodiment, L 2 =Val-Ala.

[0233] R 17 R represents an amino acid side chain, preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyl lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine and citrulline. Preferred amino acid side chains are side chains of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp and Asn, more preferably from the side chains of Val, Cit, Ala, Glu and Lys. Or in other words, R 17Preferably, it is selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred examples are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu) and CH(CH3)2(Val). Most preferably, R 17 It is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys) or CH(CH3)2(Val).

[0234] In a particularly preferred embodiment, the peptide spacer can be represented by the general structure (L3):

[0235]

[0236] In this paper, R 17 As defined above, preferably R 17 It is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates the same as (L 1 ) n and (L 3 ) p connection, preferably according to the structure (L3) 2 Through NH and (L 1 ) n Connected and connected via C(O) and (L 3 ) p connect.

[0237] Connector L 3

[0238] The linker L is absent (p=0) or present (p=1) 3 Preferably, there is a linker L 3 And p = 1. Connector L 3 is a self-cleavable spacer, also known as a self-eliminating spacer. 3is a p-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L4):

[0239]

[0240] In this article, the wavy line indicates the difference between Q or Z, L 1 or L 2 And with L 4 Or D. Usually, PABC derivatives are connected to Q, Z, L via NH. 1 or L 2 connection, preferably with L 2 connected, and through O and L 4 or D connection.

[0241] A is a 5-membered or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,4-phenyl, 2,5-pyridyl or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0242] R 21 Selected from H, R 26 , C(O)OH and C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl groups, C3–C 10 Alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally replaced by one or more selected from O, S and NR 28 The heteroatom interval, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 It is C3–C 10 (Hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or –(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably -(CH2CH2O) s H, wherein s is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s=1, 2, 3 or 4. More preferably, R 21 is H or C(O)R 26 , where R 26=4-methyl-piperazine or morpholine. Most preferably, R 21 It's H.

[0243] Connector L 4

[0244] The linker L is absent (q=0) or present (q=1) 4 Preferably, there is a linker L 4 And q = 1. Connector L 4 Selected from:

[0245] - According to the structure – NR 22 –(C z -alkylene)-C(O)-aminoalkanoic acid spacer, wherein z is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl;

[0246] - According to the structure – NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -C(O)-, wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R 22 is H or C1–C4 alkyl; and

[0247] - According to the structure – NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer, wherein h is 0 or 1, z is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl.

[0248] Connector L 4 It can be an aminoalkanoic acid spacer, i.e. –NR 22 –(C z -alkylene)-C(O)-, wherein z is an integer in the range of 1 to 20, preferably 1 to 10, most preferably 1 to 6. Herein, the aminoalkanoic acid spacer is usually attached to L via a nitrogen atom. 3 and connected to D via the carbonyl moiety. Preferred linkers L 4 is selected from 6-aminocaproic acid (Ahx, z=5), β-alanine (z=2) and glycine (Gly, z=1), even more preferably 6-aminocaproic acid or glycine. 4 =6-aminocaproic acid. In one embodiment, L 4 = glycine. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R22 It's H.

[0249] Alternatively, connector L 4 It can be based on the structure – NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -(C(O)-)ethylene glycol spacer, wherein e6 is an integer in the range of 1 to 10, preferably e6 is in the range of 2 to 6, and e7 is an integer in the range of 1 to 3, preferably e7 is 2. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 It's H.

[0250] Alternatively, connector L 4 It can be based on the structure – NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer, wherein h is 0 or 1, z is an integer in the range of 1 to 20, preferably an integer in the range of 2 to 6, even more preferably z=2 or 5, most preferably z=2. 22 is H or C1–C4 alkyl. 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 Herein, h is preferably 1, in which case the linker L 4 Particularly suitable for conjugation via the phenolic hydroxyl groups present on the payload D.

[0251] Payload D

[0252] D represents the target molecule D which is or will be linked to the antibody, which is also referred to as a payload in the art. D is exotecan.

[0253] The compounds according to the general structure (2) may contain more than one moiety D. When more than one cytotoxin D is present, the cytotoxin D may be the same or different, typically they are the same. In a preferred embodiment, the compounds according to the general structure (2) contain 1 or 2 occurrences of D, most preferably 2 occurrences of D. Typically, the second occurrence of D is present in a linker L, which may contain a branching moiety, typically a branching nitrogen atom, which is connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branching moiety via the same linker. Likewise, the antibody conjugates according to structure (1) may contain more than one D moiety per linker Z.

[0254] Preferred Antibody Conjugates

[0255] Preferred antibody conjugates according to the first aspect are selected from the group consisting of compounds (I)-(III), more preferably (II) or (III), most preferably (II). More preferred antibody conjugates are selected from (X)-(XIII). In a particularly preferred embodiment, the antibody conjugate is selected from (Xa), (XIb), (XIIg), (XIIh) and (XIIIe). In an even more preferred embodiment, the antibody conjugate is selected from (XI) and (XIII), more preferably (XIb) or (XIIIe), more preferably the antibody conjugate is according to (XIII), most preferably according to (XIIIe). The structures of these antibody conjugates are defined as follows.

[0256] The antibody conjugate (I) has the following structure:

[0257] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–(L 4 ) q –D} x ] y

[0258] (I)

[0259] in:

[0260] -AB, L 6 , Z, D, x and y are as defined above;

[0261] -L 1 is made by – (A) d –(B) e –(A) f –(C(O)) g - a linker denoted as defined above;

[0262] -L 2 It is Val-Cit or Val-Ala;

[0263] -L 3 is a PABC derivative according to structure (L4);

[0264] -L 4 Yes – N – (C z -alkylene)-C(O)- or -NR 22 –(C z -alkylene)–NR 22 –, where z and R 22 As defined above;

[0265] -q=0 or 1.

[0266] In the case of antibody conjugate (I), preferably for L 1 , d = 1 (according to A of structure (23), preferably a = 1 and R 13 =H), e=2, f=0 and g=1. In the case of antibody conjugate (I), preferably L 2 =Val-Cit. In the case of antibody conjugate (I), preferably, for L 3 , R 21 =H. In the case of the antibody conjugate (I), preferably, if q=1, then z=1 or 5.

[0267] In a particularly preferred embodiment, the antibody conjugate according to structure (I) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0268] The antibody conjugate (II) has the following structure:

[0269] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–D} x ] y

[0270] (II)

[0271] in:

[0272] -AB, L 6 , Z, D, x and y are as defined above;

[0273] -L 1 It is composed of – (A) – (B) e – a linker represented by (C(O))– is as defined above;

[0274] -L 2 It is Val-Cit or Val-Ala;

[0275] -L 3 is a PABC derivative according to structure (L4), wherein R 21 =H.

[0276] In the case of antibody conjugate (II), preferably, for L 1 , e=2, according to structure (23), preferably, a=1 and R 13 =H. In the case of antibody conjugate (II), preferably L2 =Val-Cit.

[0277] In a particularly preferred embodiment, the antibody conjugate according to structure (II) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0278] The antibody conjugate (III) has the following structure:

[0279] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–(L 4 )–D} x ] y

[0280] (III)

[0281] in:

[0282] -AB, L 6 , Z, D, x and y are as defined above;

[0283] -L 1 It is composed of – (A) – (B) e – a linker represented by (C(O))– is as defined above;

[0284] -L 2 It is Val-Cit or Val-Ala;

[0285] -L 3 is a PABC derivative according to structure (L4), wherein R 21 =H;

[0286] -L 4 Yes – NR 22 –(C z -alkylene)–NR 22 –, where R 22 As defined above and z is an integer in the range of 1 to 6.

[0287] In the case of antibody conjugate (III), preferably, for L 1 , e = 2, and a = 1 and R 13 =H. In the case of antibody conjugate (III), preferably, L 2 =Val-Cit. In the case of antibody conjugate (III), preferably z=2.

[0288] In a particularly preferred embodiment, the antibody conjugate according to structure (III) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0289] The antibody conjugate (X) has a linker payload portion according to the following structure:

[0290]

[0291] in:

[0292] - The wavy line indicates the connection to Z;

[0293] -L 2 , o and D are as defined above.

[0294] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (Xa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (Xb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (X) preferably has structure (Xa).

[0295] In a particularly preferred embodiment, the antibody conjugate according to structure (X) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0296] The antibody conjugate (XI) has a linker payload portion according to the following structure:

[0297]

[0298] in:

[0299] - The wavy line indicates the connection to Z;

[0300] -L 2 , o and D are as defined above.

[0301] L may or may not be present 2 , preferably there is L 2and o = 1. For the preferred antibody conjugate (XIa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (XI) preferably has structure (XIb).

[0302] In a particularly preferred embodiment, the antibody conjugate according to structure (XI) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably calicheamicin as the payload D.

[0303] The antibody conjugate (XII) has a linker payload portion according to the following structure:

[0304]

[0305] in:

[0306] - The wavy line indicates the connection to Z;

[0307] -L 2 , L 4 , o, q and D are as defined above.

[0308] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (XIIa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2.

[0309] L may or may not be present 4 For the preferred antibody conjugate (XIIc), q=0 and L 4 For the preferred antibody conjugate (XIId), q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R22 is H or C1–C4 alkyl.

[0310] For the preferred antibody conjugate (XIIe), L 2 According to the structure (L3), R 17 is CH3, q=0 and L 4 For the preferred antibody conjugate (XIIf), L 2 According to the structure (L3), R 17 is CH3, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 10, preferably wherein z=2, and R 22 is H or C1–C4 alkyl.

[0311] For the preferred antibody conjugate (XIIg), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0 and L 4 For the preferred antibody conjugate (XIIh), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0312] In the case of antibody conjugate (XII), preferably z is an integer in the range of 1 to 10, more preferably z=2 to 6, most preferably z=2. In the case of antibody conjugate (XII), further preferably R 22 It is H or CH3.

[0313] In the case of antibody conjugate (XII), structures (XIIg) and (XIIh) are most preferred.

[0314] In a particularly preferred embodiment, the antibody conjugate according to structure (XII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0315] The antibody conjugate (XIII) has a linker payload portion according to the following structure:

[0316]

[0317] in:

[0318] - The wavy line indicates the connection to Z;

[0319] -L 2 , L 4 , o, q and D are as defined above.

[0320] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (XIIIa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIIIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH3.

[0321] L may or may not be present 4 For the preferred antibody conjugate (XIIIc), q=0 and L 4 For the preferred antibody conjugate (XIIId), q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0322] For the preferred antibody conjugate (XIIIe), L 2 According to the structure (L3), R 17 is CH3, q=0 and L 4 For the preferred antibody conjugate (XIIIf), L 2 According to the structure (L3), R 17 is CH3, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 10, preferably wherein z=2, and R 22 is H or C1–C4 alkyl.

[0323] For the preferred antibody conjugate (XIIIg), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0 and L 4 For the preferred antibody conjugate (XIIIh), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0324] In the case of antibody conjugate (XIII), preferably z is an integer in the range of 1 to 10, more preferably z=2 to 6, most preferably z=2. In the case of antibody conjugate (XIII), further preferably R 22 It is H or CH3.

[0325] In the case of antibody conjugate (XIII), structure (XIIIe) is most preferred.

[0326] In a particularly preferred embodiment, the antibody conjugate according to structure (XIII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0327] In a particularly preferred embodiment the antibody conjugate according to the invention is according to structure (XIIIe) as defined above, and the antibody is hRS7 as defined above and the payload is exotecan.

[0328] It is further preferred that these preferred antibody conjugates are conjugated via a glycan, i.e. b=1, more preferably a trimmed glycan, i.e. j=0. Here, it is further preferred that S=GalNAc and w'=0. Here, it is further preferred that the linker Z is formed by an azide-alkyne cycloaddition, preferably the linker Z=(Z39), wherein ring Z=(Za) and V=CH2. Here, it is further preferred that x=1. Here, it is further preferred that y=2, more preferably x=1 and y=2.

[0329] In the most preferred embodiment, the antibody conjugate according to the present invention is according to structure (XIIIe) as defined above, and the antibody is hRS7 as defined above and the payload is exotecan, wherein b=1, e=0, S=GalNAc, w'=0, linker Z=(Z39), wherein ring Z=(Za) and V=CH2, x=1 and y=2.

[0330] Compounds according to formula (2)

[0331] The compound has the general structure (2):

[0332] Q–L–D (2)

[0334] in:

[0335] -Q is the reactive moiety;

[0336] -L is the linker connecting Z to D;

[0337] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitin, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, indoline benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0338] The compound of general structure (2) may also be referred to as a "linker-drug construct" for comprising the linker L and the payload D of the final conjugate. The compound according to general formula (2) can be prepared by a person skilled in the art using standard organic synthesis techniques and as illustrated in the Examples. The linker L and the payload D are defined in the context of the conjugate of structure (1).

[0339] Reactive part Q

[0340] The compounds according to the general structure (2) contain a reactive moiety Q. In the context of the present invention, the term "reactive moiety" may refer to a chemical moiety containing a reactive group, but may also refer to the reactive group itself. For example, a cyclooctynyl group is a reactive group containing a reactive group, namely a C-C triple bond. Similarly, an N-maleimido group is a reactive group containing a C-C double bond as a reactive group. However, reactive groups, such as an azide reactive group, a sulfhydryl reactive group or an alkynyl reactive group, may also be referred to herein as a reactive moiety.

[0341] Q is used to connect to S(F) xIn other words, Q is reactive to F and complementary to F. Herein, a reactive group is referred to as being "complementary" to a reactive group when the reactive group selectively reacts with the reactive group, optionally in the presence of other functional groups. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Thus, compounds according to general structure (2) are conveniently used in a conjugation reaction, wherein a chemical reaction occurs between Q and F to form an antibody conjugate comprising a covalent bond between the payload D and the antibody.

[0342] The exact nature of Q and F depends on the type of conjugation reaction used. Those skilled in the art will be able to select an appropriate combination of Q and F. Preferably, Q and F are reactive in cycloadditions or nucleophilic reactions. Therefore, Q preferably comprises a click probe, a sulfhydryl, a sulfhydryl reactive moiety, an amine or an amine reactive moiety, more preferably Q is a click probe, a sulfhydryl reactive moiety or an amine reactive moiety, and most preferably Q is a click probe. The click probe is reactive in cycloadditions (click reactions) and is preferably selected from azides, tetrazines, triazines, nitrone, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxythiophenes, Sydney ketones, olefin moieties and alkyne moieties. Preferably, the click probe comprises or is an olefin moiety or an alkyne moiety, more preferably wherein the olefin is a (hetero) cycloolefin and / or the alkyne is a terminal alkyne or a (hetero) cycloalkyne. Typical sulfhydryl reactive moieties are selected from maleimide moieties, haloacetamide moieties, allenamide moieties, phosphinamide moieties, cyanoacetylene moieties, vinyl sulfone, vinyl pyridine moieties or methylsulfonylphenyl oxadiazole moieties. Most preferably, the sulfhydryl reactive moieties include or are maleimide moieties. Typical amine reactive moieties are selected from N-hydroxysuccinimide esters and other activated esters, p-nitrophenyl carbonate and other activated carbonates, isocyanates, isothiocyanates, haloacetamides and benzyl halides. In a preferred embodiment, Q is selected from olefin moieties, alkyne moieties, sulfhydryl reactive moieties or amine reactive moieties, more preferably olefin moieties or alkyne moieties, even more preferably alkyne moieties. Herein, olefin is preferably (hetero) cycloolefin, and alkyne is preferably terminal alkyne or (hetero) cycloalkyne. Most preferably, Q is a cyclic (hetero) alkyne moiety. Each of these moieties is further defined below.

[0343] Therefore, in a particularly preferred embodiment, Q comprises a cyclic (hetero) alkynyl moiety.Alkynyl groups may also be referred to as (hetero) cycloalkynyl groups, i.e. heterocycloalkynyl groups or cycloalkynyl groups, wherein the (hetero) cycloalkynyl groups are optionally substituted.Preferably, the (hetero) cycloalkynyl groups are (hetero) cycloheptynyl groups, (hetero) cyclooctynyl groups, (hetero) cyclononyl groups or (hetero) cyclodecynyl groups.Herein, (hetero) cycloalkynes may be optionally substituted.Preferably, the (hetero) cycloalkynyl groups are optionally substituted (hetero) cycloheptynyl groups or optionally substituted (hetero) cyclooctynyl groups.Most preferably, the (hetero) cycloalkynyl groups are (hetero) cyclooctynyl groups, wherein the (hetero) cyclooctynyl groups are optionally substituted.

[0344] In a particularly preferred embodiment, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and is according to structure (Q1):

[0345]

[0346] In this article:

[0347] -Depicted as The bond is a double bond or a triple bond;

[0348] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0349] –C 24 The group consisting of (hetero)arylalkyl groups;

[0350] -Y 2 is C(R 31 )2. O. S. S (+) R 31 、S(O)R31 、S(O)=NR 31 or NR 31 , where S (+) It was B (-) The balanced cationic sulfur atom, where B (-) is an anion, and each R 31 R alone 15 or a connection to D, which is connected via L;

[0351] -u is 0, 1, 2, 3, 4, or 5;

[0352] -u' is 0, 1, 2, 3, 4 or 5, wherein u+u'=0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0353] -v=an integer in the range of 0 to 16;

[0354] Usually, v = (u + u') x 2 (when passing through Y 2 When connected to L (represented by a wavy bond)) or [(u+u')x 2]-1 (when connected to L via one of the carbon atoms (represented by a wavy bond)).

[0355] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1a):

[0356]

[0357] In this article,

[0358] -R 15 and Y 2 As defined above

[0359] -u is 0, 1, 2, 3, 4, or 5;

[0360] -u' is 0, 1, 2, 3, 4 or 5, where u+u'=4, 5, 6, 7 or 8;

[0361] -v=an integer in the range of 8 to 16.

[0362] In a preferred embodiment, u+u'=4, 5 or 6, more preferably u+u'=5. In a preferred embodiment, v=8, 9 or 10, more preferably v=9 or 10, most preferably v=10.

[0363] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) - (Q20) and (Q20a) depicted below.

[0364]

[0365] Herein, the connection to L depicted by the wavy bond can be a connection to any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) can have a connection to L, or can contain a hydrogen atom or be optionally functionalized. (–) is an anion, which is preferably selected from (–) OTf、Cl (–) Br (–) or I (–) , most preferably B (–) yes (–) OTf. In the conjugation reaction, B (–) does not need to be a pharmaceutically acceptable anion, because B (–) In any case, exchange will take place with anions present in the reaction mixture. In case (Q19) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable when isolating the conjugate according to the invention, making the conjugate easy to use as a drug.

[0366] In further preferred embodiments, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21)-(Q38) and (Q38a) depicted below.

[0367]

[0368] In structure (Q38), B (–) is an anion, which is preferably selected from (–) OTf、Cl (–) Br (–) or I (–) , most preferably B (–) yes (–) OTf.

[0369] In a preferred embodiment, Q comprises a (hetero)cyclooctyne moiety or a (hetero)cycloheptyne moiety, preferably according to Structure (Q8), (Q26), (Q27), (Q28), (Q37) or (Q38a), more preferably according to Structure (Q8), (Q26), (Q27), (Q28) or (Q37), which is optionally substituted. Each of these preferred options of Q is further defined below.

[0370] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is unsubstituted.

[0371] In alternative preferred embodiments, Q comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also known as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q39) as shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q is structure (Q42) as further defined below.

[0372] In alternative preferred embodiments, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also known as a DIBO, DIBAC, DBCO or ADIBO group, which is optionally substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 are independently selected from hydrogen, a linear or branched C1-C 12 Alkyl group or C4-C 12 The aromatic ring in (Q40) is optionally O-sulfonylated at one or more positions, while the ring of (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is structure (Q43) as further defined below.

[0373] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is according to structure (Q37).

[0374]

[0375] In a particularly preferred embodiment, Q comprises a cyclooctynyl group and is according to structure (Q42):

[0376]

[0377] In this article:

[0378] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3(-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alk(hetero)aryl and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–

[0379] C 24 The group consisting of (hetero)arylalkyl groups;

[0380] -R 18 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups;

[0381] -R 19 Selected from hydrogen, halogen, C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, the alkyl groups are optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Q or D linked via a spacer moiety; and

[0382] -l is an integer in the range 0 to 10.

[0383] In a preferred embodiment of the reactive group according to Structure (Q42), R 15 are independently selected from hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In a preferred embodiment of the reactive group according to Structure (Q42), R 18 Independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably two R 18 are all H. In a preferred embodiment of the reactive group according to Structure (Q42), R 19 is H. In a preferred embodiment of the reactive group according to Structure (Q42), l is 0 or 1, more preferably l is 1.

[0384] In a particularly preferred embodiment, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43):

[0385]

[0386] In this article:

[0387] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl(hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein the alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 are independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0388] –C 24 The group consisting of (hetero)arylalkyl groups;

[0389] -Y is N or CR 15 .

[0390] In a preferred embodiment of the reactive group according to Structure (Q43), R 15 are independently selected from hydrogen, halogen, -OR 16 、-S(O)3(-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from hydrogen and -S(O)3 (-) In a preferred embodiment of the reactive group according to Structure (Q43), Y is N or CH, more preferably Y=N.

[0391] In particularly preferred embodiments, Q comprises a heterocycloheptenyl group and is according to structure (Q37) or (Q38a), preferably according to structure (Q37)

[0392]

[0393] In an alternative preferred embodiment, Q comprises a cycloolefin moiety.Alkenyl Q may also be referred to as a (hetero)cycloalkenyl group, i.e. a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cycloalkenyl group is optionally substituted.Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornenyl group, a norbornadienyl group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted.Particularly preferred are a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group or a trans-(hetero)cyclooctenyl group, wherein the (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group or a trans-(hetero)cyclooctenyl group are optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). Here, Y 3 Selected from C(R 23 )2. NR 23 or O, where each R 23 Each is hydrogen, C1-C6 alkyl or optionally connected to L via a spacer, and is marked as ––– The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropenyl group is according to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctenene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).

[0394]

[0395] Here, the R group on Si in (Q50) and (Q51) is generally an alkyl group or an aryl group, preferably a C1-C6 alkyl group.

[0396] In another preferred embodiment, Q is a sulfhydryl reactive probe. In this embodiment, Q is a reactive group compatible with cysteine ​​conjugation. Such probes are known in the art and can be selected from the group consisting of a maleimide moiety, a haloacetamide moiety, a propadienamide moiety, a phosphinamide moiety, a cyanoethynyl moiety, a vinyl sulfone, a vinyl pyridine moiety or a methylsulfonylphenyl oxadiazole moiety. Most preferably, Q comprises or is a maleimide moiety. Reagents can be monoalkylated types or can be cross-linking agents for reacting with two cysteine ​​side chains.

[0397] In a further preferred embodiment, probe Q is selected from the group consisting of (Q57)-(Q71) depicted below.

[0398]

[0399] in:

[0400] -X 6 is H, halogen, PhS, MeS, preferably halogen, such as Cl, Br, I;

[0401] -X 7 is halogen, PhS, MeS, preferably halogen, such as Cl, Br, I;

[0402] -R 24 Is H or C 1-12 Alkyl, preferably H or C 1-6 alkyl;

[0403] -R 25 It is H, C 1-12 Alkyl group, C 1-12 Aryl, C 1-12 Alkyl or C 1-12 Aralkyl, preferably H or p-methylphenyl;

[0404] - wherein the aromatic rings of (Q61) and (Q63) may optionally be heteroaromatic rings, such as phenyl or pyridine rings.

[0405] In a preferred embodiment of the thiol-reactive probe (Q57), the probe Q is selected from the group consisting of (Q72)-(Q74) depicted below.

[0406]

[0407] in:

[0408] -R 27 It is C1-12 Alkyl group, C 1-12 Aryl, C 1-12 Alkyl or C 1-12 Aralkyl;

[0409] -t is an integer in the range of 0-15, preferably 1-10.

[0410] In another preferred embodiment, Q is an amine reactive probe. In this embodiment, Q is a reactive group compatible with lysine conjugation. Such probes are known in the art and can be selected from the group consisting of N-hydroxysuccinimide groups, isocyanate groups, isothiocyanate groups and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimide ester or p-nitrophenyl carbonate moiety.

[0411] In a further preferred embodiment, probe Q is selected from the group consisting of (Q75) - (Q79) depicted below.

[0412]

[0413] Among them, X 2 is halogen, preferably F.

[0414] In a preferred embodiment, Q is selected from the group consisting of (Q1)-(Q79).

[0415] Antibodies according to the general structure (3)

[0416] The antibody has the general structure (3):

[0417] AB–[(L 6 ) b –{F} x ] y (3)

[0419] in:

[0420] - AB is an antibody capable of targeting tumors expressing Trop-2;

[0421] -b is 0 or 1;

[0422] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w '-, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0423] -F is the reactive moiety;

[0424] -x is 1 or 2; and

[0425] -y is 1, 2, 3, or 4.

[0426] The antibody of general structure (3) may also be referred to as a "(modified) antibody" because it is an antibody containing a reactive group F, wherein the reactive group F is naturally present or the antibody is modified to incorporate the reactive group F. The (modified) antibody according to general formula (2) can be prepared by a person skilled in the art using standard organic and / or enzymatic synthesis techniques and as illustrated in the Examples. Antibody AB, Linker L 6 , b, x and y are defined above in the context of the conjugate according to structure (1).

[0427] Reactive part F

[0428] F is reactive to Q in a conjugation reaction defined below, preferably wherein the conjugation reaction is a cycloaddition or a nucleophilic reaction. As will be appreciated by those skilled in the art, the options for F are the same as those for Q, provided that F and Q react with each other. Thus, F preferably comprises a click probe, a sulfhydryl, a sulfhydryl reactive moiety, an amine or an amine reactive moiety, more preferably F is a click probe, a sulfhydryl or an amine, most preferably F is a click probe. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene, Sydney ketone, olefin moiety and alkyne moiety. Preferably, the click probe comprises or is azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene or Sydney ketone, most preferably azide. Typical sulfhydryl reactive moieties are selected from maleimide moieties, haloacetamide moieties, propadienamide moieties, phosphinamide moieties, cyanoethynyl moieties, o-quinone moieties, vinyl sulfones, vinyl pyridine moieties, or methylsulfonylphenyl oxadiazole moieties. Most preferably, the sulfhydryl reactive moiety comprises or is a maleimide moiety. Typical amine reactive moieties are selected from N-hydroxysuccinimide esters, isocyanates, isothiocyanates, and benzyl halides. In a preferred embodiment, F is a click probe or a sulfhydryl group, more preferably F is an azide or a sulfhydryl group, and most preferably F is an azide.

[0429] More than one reactive group F may be present in an antibody. The reactive group F in an antibody may be naturally present or may be placed in the antibody by specific techniques such as (bio) chemistry or genetic techniques. The reactive group placed in the antibody is prepared by chemical synthesis, such as an azide or a terminal alkyne. Methods for preparing modified antibodies are known in the art, for example, from WO 2014 / 065661, WO 2016 / 170186 and WO 2016 / 053107, which are incorporated herein by reference. From the same literature, the conjugation reaction between the modified antibody and the linker-drug construct is known to those skilled in the art.

[0430] Preferably, F is a click probe reactive to (hetero)cycloolefins and / or (hetero)cycloalkynes, and is typically selected from the group consisting of azides, tetrazines, triazines, nitrone oxides, nitrile imines, diazo compounds, o-quinones, dioxythiophenes, and Sydney ketones. Preferred structures of the reactive group are structures (F1)-(F10) depicted below.

[0431]

[0432] Here, the wavy bond represents the connection to the payload. For (F3), (F4), (F8), and (F9), the payload can be connected to any of the wavy bonds. Then another wavy bond can be connected to a selected from hydrogen, C1–C 24 Alkyl groups, C2–C 24 Acyl group, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups, C3–C 24 (Hetero)arylalkyl groups and C1–C 24 The R group of the sulfonyl group, each of which (except hydrogen) may be optionally substituted and optionally replaced by one or more selected from O, S and NR 32 The heteroatom interval, where R 32 Independently selected from the group consisting of hydrogen and C1-C4 alkyl. Those skilled in the art understand which R groups are applicable to each group F. For example, the R group attached to the nitrogen atom of (F3) can be selected from alkyl and aryl, and the R group attached to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl and sulfonyl. Preferably, the reactive moiety F is selected from azide or tetrazine. Most preferably, the reactive moiety F is azide.

[0433] In a second preferred embodiment, F is a thiol group or a precursor thereof. The thiol group or its precursor F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F reacts with the thiol-reactive probe Q in the thiol connection. The thiol group is preferably a thiol group of the side chain of cysteine ​​amino acid, which naturally occurs in the antibody AB, in which case the linker L is not present. 6 (b = 0), but it can also optionally be connected via a linker L 6 Thiol precursors in the context of bioconjugation are known in the art and include disulfide bonds, which may be naturally occurring disulfide bonds present in antibodies or synthetically introduced disulfide bonds, which are reduced as known in the art. Preferably, F is a thiol group of a cysteine ​​side chain.

[0434] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or its precursor F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F reacts with the amine-reactive probe Q in a nucleophilic substitution. The amine is typically a primary amine, preferably an amine of the side chain of a lysine amino acid, which is naturally present in the antibody AB, in which case the linker L is absent. 6 (b = 0), but it can also optionally be connected via a linker L 6 Preferably, F is a primary amine group of a lysine side chain.

[0435] Method for synthesizing antibody conjugates according to general structure (1)

[0436] On the other hand, the present invention relates to a method for preparing an antibody conjugate according to the present invention, the method comprising the step of reacting Q of a compound according to the present invention with a reactive group F of an antibody. Compounds according to the general structure (2) and preferred embodiments thereof are described in more detail above. The method occurs under the conditions that Q reacts with F to covalently link the antibody AB (3) to a payload D. In the method according to the present invention, Q reacts with F to form a covalent connection between the antibody and the compound according to the present invention. Complementary reactive groups Q and reactive groups F are known to those skilled in the art and are described in more detail below.

[0437] Any conjugation technique known in the art can be used to prepare the multifunctional antibody construct according to the present invention. Suitable conjugation techniques include sulfhydryl linkage, lysine linkage, cycloaddition (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cycloaddition, strain-promoted quinone-alkyne cycloaddition). Preferred conjugation techniques used in the context of the present invention include nucleophilic reactions and cycloadditions, preferably wherein the cycloaddition is a [4+2] cycloaddition or a [3+2] cycloaddition and the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Suitable conjugation techniques are disclosed in, for example, GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), WO 2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233–2242, PCT / EP2021 / 050594, PCT / EP2021 / 050598 and NL2026947.

[0438] Therefore, in a preferred embodiment of the conjugation method according to the present invention, the conjugation is accomplished via a nucleophilic reaction such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is acylation of the primary amino group with an activated ester. A preferred Michael reaction is a maleimide-thiol reaction, which is widely used in bioconjugation.

[0439] Therefore, in a preferred embodiment of the conjugation method according to the present invention, conjugation is completed via cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reactions) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition reaction. Preferred Diels-Alder reactions are reverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, 1,3-dipolar cycloadditions are used, more preferably alkyne-azide cycloadditions, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and those skilled in the art know how to perform them.

[0440] The method according to this aspect preferably involves a click reaction, more preferably a 1,3-dipolar cycloaddition, most preferably an alkyne / azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azide group. Click reactions, such as 1,3-dipolar cycloadditions, are known in the art and those skilled in the art know how to perform them.

[0441] Thus, according to this aspect, the method for preparing an antibody conjugate according to the invention comprises reacting a modified antibody of structure (3) with a compound according to structure (2) to obtain an antibody conjugate of structure (1).

[0442] In a preferred embodiment, the method for preparing the antibody conjugate according to the present invention comprises:

[0443] (i) combining an antibody comprising a y-core N-acetylglucosamine (GlcNAc) portion with a compound of formula S(F) x -P compound in the presence of a catalyst, wherein y = 1, 2, 3 or 4, wherein S (F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of reacting S(F) x The core-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26):

[0444] AB–[GlcNAc(Fuc) w –S{F} x ] y (26)

[0446] in

[0447] - AB is an antibody capable of targeting tumors expressing Trop-2;

[0448] -Fuc is fucose;

[0449] -w is 0 or 1; and

[0450] (ii) reacting the modified antibody with a compound according to structure (2):

[0451] Q–L–D (2)

[0453] in:

[0454] -Q is the reactive moiety;

[0455] -L is the linker connecting Z to D;

[0456] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitin, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, indoline benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0457] to obtain an antibody conjugate according to structure (1).

[0458] Step (i)

[0459] In step (i), an antibody comprising 1, 2, 3 or 4 core N-acetylglucosamine moieties is combined with a x -P compound in the presence of a catalyst, wherein S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with a reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The antibody is typically one that has been trimmed to core-GlcNAc residues, as further described below. Step (i) provides a modified antibody according to formula (26).

[0460] Starting material, i.e., antibodies comprising core-GlcNAc substituents, are known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the method according to the present invention also includes deglycosylation of antibody polysaccharides with core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the polysaccharide, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and / or a combination thereof, and its selection depends on the nature of the polysaccharide. EndoSH is described in PCT / EP2017 / 052792, see Examples 1 to 3 and SEQ.ID No: 1, which is incorporated herein by reference.

[0461] The structural features S and x are defined above for the antibody conjugates according to the invention and are also applicable to the present invention. x Connection type S(F) x -P compounds are known in the art. For example, Wang et al., Chem. Eur. J. 2010, 16, 13343-13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462 and WO 2009 / 102820, all of which are incorporated herein by reference, disclose many compounds S (F)x -P and its synthesis. In a preferred embodiment, S(F) x -The nucleoside monophosphate or diphosphate P in P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP), most preferably P = UDP. Preferably, S (F) x -P is selected from the group consisting of GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetylgalactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-ynoic acid amide)-2-deoxy-galactose-UDP. Most preferably, S(F) x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0462] Can S(F) x Suitable catalysts for the transfer of moieties to the core-GlcNAc moiety are known in the art. Suitable catalysts are catalysts for which the specific sugar derivative nucleotide S(F) in this specific method x-P is a substrate. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group consisting of galactosyltransferase and N-acetylgalactosaminyltransferase, more preferably selected from the group consisting of β(1,4)-N-acetylgalactosaminyltransferase (GalNAcT) and β(1,4)-galactosaminyltransferase (GalT), most preferably selected from the group consisting of β(1,4)-N-acetylgalactosaminyltransferase with a mutant catalytic domain. Suitable catalysts and mutants thereof are disclosed in WO 2014 / 065661, WO 2016 / 022027, and WO 2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. Particularly preferred are the mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186. These galactosyltransferase (mutant) enzyme catalysts are able to recognize internal sugars and sugar derivatives as acceptors. Thus, the sugar derivative S (F) x In step (i) a core-GlcNAc substituent is attached, whether or not said GlcNAc is fucosylated.

[0463] Step (i) is preferably carried out in a suitable buffer solution, such as, for example, phosphate, buffered saline (e.g., phosphate buffered saline, tris buffered saline), citrate, HEPES, tris and glycine. Suitable buffers are known in the art. Preferably, the buffer solution is phosphate buffered saline (PBS) or tris buffer. Step (i) is preferably carried out at a temperature in the range of about 4 to about 50°C, more preferably in the range of about 10 to about 45°C, even more preferably in the range of about 20 to about 40°C and most preferably in the range of about 30 to about 37°C. Step (i) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (i) is carried out at a pH in the range of about 7 to about 8.

[0464] Step (ii)

[0465] In step (ii), the modified antibody is reacted with a compound according to general structure (2) to obtain an antibody conjugate according to structure (1), the compound comprising a reactive group Q capable of reacting with a reactive group F and a payload D, the antibody conjugate containing a linker group Z resulting from the reaction between Q and F. This reaction occurs under conditions where the reactive group Q reacts with the reactive group F of the biomolecule to covalently link the antibody to the compound according to general structure (2). Step (ii) may also be referred to as a conjugation reaction.

[0466] In a preferred embodiment, in step (ii), the azide on the azide-modified antibody reacts with an alkynyl group, preferably a terminal alkynyl group or a (hetero)cycloalkynyl group of a compound according to general structure (2) via a cycloaddition reaction. This cycloaddition reaction of a molecule comprising an azide with a molecule comprising a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the "click chemistry" reactions known in the art. In the case of a linker-conjugate comprising a terminal alkynyl group, the cycloaddition reaction needs to be carried out in the presence of a suitable catalyst, preferably a Cu(I) catalyst. However, in a preferred embodiment, the linker-conjugate comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl group is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required, and the reaction can even occur spontaneously through a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry".

[0467] application

[0468] The present invention further relates to a method for treating a subject in need thereof, comprising administering an antibody conjugate according to the present invention as defined above. A subject in need thereof is typically a cancer patient. The use of antibody conjugates, such as antibody-drug conjugates, in the field of cancer treatment is well known, and the antibody conjugates according to the present invention are particularly suitable for this aspect. The described method is generally suitable for the treatment of cancer. In the method according to this aspect, the antibody conjugate is generally administered in a therapeutically effective dose. This aspect of the present invention can also be expressed as an antibody conjugate according to the present invention, which is used to treat a subject in need thereof, preferably for the treatment of cancer. In other words, this aspect relates to the use of an antibody conjugate according to the present invention for the preparation of a medicament or a pharmaceutical composition, which is used to treat a subject in need thereof, preferably for the treatment of cancer. In this article, the treatment of cancer is expected to include treating, imaging, diagnosing, preventing the proliferation of tumors, inhibiting and reducing tumors.

[0469] This aspect of the invention may also be formulated as a method for targeting cells expressing Trop-2, in particular Trop-2 expressing tumor cells, comprising contacting an antibody conjugate according to the invention with cells that may be Trop-2 expressing. Thus, the method according to this aspect is suitable for determining whether a cell expresses Trop-2. These Trop-2 expressing cells may be present in a subject, in which case the method comprises administering an antibody conjugate according to the invention to a subject in need thereof. In a preferred embodiment, the cells that may be Trop-2 expressing are Trop-2 expressing cells. Targeting cells that express Trop-2 preferably comprises one or more of the following: treating, imaging, diagnosing, preventing proliferation of, inhibiting and reducing Trop-2 expressing cells, in particular Trop-2 expressing tumor cells. The method according to this embodiment may be medical or non-medical. The non-medical method according to this aspect may involve targeting cells that express Trop-2 in vitro or ex vivo, wherein the cells that may be Trop-2 expressing are present in a sample, such as a sample taken from a patient. Such non-medical methods are typically used for diagnosing cancer, in particular Trop-2 positive cancer.

[0470] In the context of the present invention, the subject may be suffering from a condition selected from oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer and lung cancer. Therefore, the treatment of a subject in need preferably refers to the treatment of oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer and lung cancer.

[0471] The inventors surprisingly found that the antibody conjugates according to the present invention are superior to conventional Trop-2 targeting antibody conjugates in terms of safety and / or efficacy, such that the therapeutic index of the antibody conjugates according to the present invention is increased relative to conventional Trop-2 targeting antibody conjugates.

[0472] Conjugation mode

[0473] In the context of the present invention, "conjugation mode" refers to the method used to conjugate the payload D to the antibody AB, as well as the structural features of the resulting antibody conjugate, in particular the structural features of the linker connecting the payload to the antibody, which are a direct result of the conjugation method. Therefore, in one embodiment, the conjugation mode refers to the method of conjugating the payload to the antibody. In an alternative embodiment, the conjugation mode refers to the structural features of the linker and / or the point of attachment of the linker to the antibody, which are a direct result of the method of conjugating the payload to the antibody.

[0474] In another aspect, the present invention relates to the use of a conjugation mode for increasing the therapeutic index of an antibody conjugate in the treatment of a tumor expressing Trop-2, wherein the conjugation mode is used to connect the antibody AB to the payload D via a linker L. The conjugation mode includes:

[0475] (i) combining an antibody comprising a y-core N-acetylglucosamine (GlcNAc) portion with a compound of formula S(F) x -P compounds in the presence of catalyst

[0476] Contact below, where y = 1, 2, 3 or 4, where S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of reacting S(F) x The core-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26):

[0477] AB–[GlcNAc(Fuc) w –S{F} x ] y (26)

[0479] in

[0480] - AB is an antibody capable of targeting tumors expressing Trop-2;

[0481] -Fuc is fucose;

[0482] -w is 0 or 1; and

[0483] (ii) reacting the modified antibody with a compound according to structure (2):

[0484] Q–L–D (2)

[0486] in:

[0487] -Q is the reactive moiety;

[0488] -L is the linker connecting Z to D;

[0489] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitin, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, indoline benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0490] Obtaining an antibody conjugate according to structure (1)

[0491] Preferably, increasing the therapeutic index of the antibody conjugate is selected from:

[0492] (a) improving the therapeutic efficacy of the antibody conjugate; and / or

[0493] (b) Increasing the tolerability of antibody conjugates.

[0494] The increase in therapeutic efficacy of the antibody conjugates according to the invention may take the form of a reduction in tumor size and / or a prolonged regression time when compared to conventional ADCs targeting Trop-2. The increase in tolerability of the antibody conjugates according to the invention may take the form of a reduction in signs of toxicity compared to administration of ADCs targeting Trop-2 prepared using conventional techniques. The reduction in symptoms may also be referred to as a reduction in symptoms or side effects of cancer treatment, and may involve one or more clinical signs, such as a reduction in weight loss, a reduction in decreased mobility, a reduction in decreased food intake, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology.

[0495] Examples

[0496] General Procedure for Transient Expression and Purification of Monoclonal Antibodies :Evitria (Zurich, Switzerland) transiently expressed various IgG (hRS7 and hTINA) in CHO K1 cells with 300mL scale. Use HiTrap MabSelect Sure column to purify supernatant. The supernatant is loaded onto the column and then washed with 25mM Tris pH 7.5, 150mM NaCl (TBS) of at least 10 times of column volume. The retained protein is eluted with 0.1M AcOH pH 2.7. Immediately neutralize the eluted product with 2.5M Tris-HCl pH 8.8, and dialyze with 20mM histidine, 150mM NaCl, pH 7.5. Next, Vivaspin Turbo 15 ultrafiltration devices (Sartorius) are used to concentrate IgG (> 20mg / mL). The sequence of IgG is given below:

[0497] hRS7(I) light chain (SEQ ID No.3):

[0498] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPD

[0499] RFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSD

[0500] EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0501] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0502] hRS7(I) heavy chain (SEQ ID No.4):

[0503] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0504] hTINA(II) light chain (SEQ ID No.5):

[0505] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0506] hTINA(II) heavy chain (SEQ ID No.6):

[0507] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0508] General Procedure for Analytical RP-UPLC (DTT-treated samples) : Before RP-UPLC analysis, IgG (10 μL, 1 mg / mL, dissolved in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 μL) and incubated at 37°C for 15 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on WatersAcquity UPLC-SQD. The sample (5 μL) was injected into Bioresolve RP mAb 2.1*150mm 2.7μm (Waters) at a speed of 0.4 mL / min, and the column temperature was 70°C. A linear gradient of 30% to 54% acetonitrile in 0.1% TFA and water was applied in 9 minutes. The absorbance of the elution peak was measured at 215 nm, and then automatic integration (MassLynx, Waters) was performed to determine the reaction conversion.

[0509] Mass spectrometry analysis of (modified) monoclonal antibodiesGeneral procedure: Prior to mass spectrometry analysis, IgG was treated with IdeS so that the Fc / 2 fragment could be analyzed. For the analysis of light and heavy chains, a 20 μg solution of (modified) IgG was incubated with 100 mM DTT (total volume was 4 μL) at 37°C for 5 minutes. If present, the azide function was reduced to an amine under these conditions. For the analysis of Fc / 2 fragments, a 20 μg solution of (modified) IgG was incubated with IdeS / FabricatorTM (1.25 U / μL) in phosphate buffered saline (PBS) pH 6.6 (total volume was 10 μL) at 37°C for 1 hour. The sample was diluted to 80 μL and then subjected to electrospray ionization time-of-flight (ESI-TOF) analysis on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0510] General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg / mL) was mixed with 1% w / w EndoSH (as described in PCT / EP2017 / 052792, see Examples 1-3, and SEQ. ID No: 1, which is incorporated herein by reference), 3% w / w His-TnGalNAcT (as described in PCT / EP2016 / 059194, see Examples 3 and 4, and SEQ. ID No: 49, which is incorporated herein by reference), 0.01% AP (Roche) and UDP 6-N3-GalNAc ( Figure 3 Compound 2d in (25eq compared to IgG) was incubated together at 30 ° C for 16 hours in 6mM MnCl2 and TBS. Next, functionalized IgG was purified using HiTrapMabSelect sure 5mL column. After loading the reaction mixture, the column was washed with TBS+0.2% triton and TBS. IgG was eluted with 0.1M AcOH pH 2.7 and neutralized with 2.5MTris-HCl pH 8.8. After dialysis 3 times with 20mM histidine, 150mM NaCl pH 7.5, IgG was concentrated to 15-20mg / mL using Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0511] Preparation of Azide-Functionalized Antibodies:

[0512] Example 1: Preparation of hRS7-(6-N3-GalNAc)2(I-N3)

[0513] According to the general procedure of enzymatic remodeling, hRS7 was converted to hRS7-(6-N3-GalNAc)2. Mass spectrometry analysis of samples after IdeS treatment showed a major Fc / 2 product (observed mass 24360 Da, approximately 80% of total Fc / 2), corresponding to the expected product.

[0514] Examples 2 to 4: Synthesis of Linker Conjugates 3 and 9

[0515]

[0516] Example 2: Preparation of Compound 11

[0517] Compound 10 (163 mg, 240 μmol) was added to a mixture of exitecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in anhydrous DMF (0.9 mL). After 20 h, the reaction mixture was diluted to 9 mL DCM and purified by gradient column chromatography (0→40% MeOH / DCM) to give 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) calculated value C 55 H 54 F6O 10 + (M+H) + 977.39, found 977.72. In addition to 11, ixotecan free base (82.4 mg, 189 μmol, 20%) was also recovered. LCMS (ESI+) calculated value C 24 H 23 FN3O4 + (M+H) + 436.46, measured value 436.54.

[0518] Example 3. Preparation of Compound 3

[0519] The synthesis of BCN-HS-(va-PABC-Ex)2 (3) is also described in PCT / EP2021 / 075401 (Example 4), which is incorporated herein. To a solution of compound 11 (155 mg, 159 μmol) in DMF (1.6 mL) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 12 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL) and purified by gradient column chromatography (0→40% MeOH / DCM) to give 3 as a light yellow solid (94 mg, 44 μmol, 28%). 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + LCMS (ESI+) calcd. 1066.88, found 1067.12.

[0520]

[0521] Example 4. Preparation of Compound 9

[0522] A solution of BCN-HS-PEG2-b-(Glu(OFm)-OH)2 (8, 12.1 mg, 10 μmol, 1.0 eq) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-isotecan (5b, Fmoc deprotection 5, 19 mg, 25 μmol, 2.5 eq) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 eq) and HATU (8.9 mg, 23 μmol, 2.3 eq). After stirring at room temperature for 2 h, the reaction mixture was further diluted with DCM (800 μL) and purified by silica gel flash column chromatography (0% → 20% MeOH / DCM) to give a clear oily product (the yield was difficult to determine due to the presence of DMF). LCMS (ESI+) calculated value C 140 H 150 F2N 17 O 33 S + (M / 2+H + )1334.01, measured value 1334.79.

[0523] The compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 eq) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel flash column chromatography (0% → 45% MeOH / DCM) to afford compound 9 in 44% yield as a yellow solid (10.2 mg, 4.4 μL). LCMS (ESI+) calculated value C 112 H 130 F2N 17 O 33 S + (M / 2+H + )1156.2, measured value 1156.74.

[0524] Examples 5-7: Conjugation of linker payloads to (modified) monoclonal antibodies

[0525] Example 5: Conjugation of hRS7(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PABC-Ex)23 to obtain the conjugate hRS7-3

[0526] Sodium deoxycholate (150 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)2 3 (50 μL, 10 mM DMF solution) and propylene glycol (400 μL) were added to a solution of hRS7 (6-N3-GalNAc) 2 (764.5 μL, 15.0 mg, 19.62 mg / ml in TBS, pH 7.5). The reactants were incubated overnight at room temperature. To remove excess free payload, 15 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated for 2 hours. The charcoal was removed by centrifugation and then filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with PBS. Since the free payload level was still too high, an additional 10 mg of activated charcoal was added and incubated overnight. The charcoal was removed by centrifugation and then filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20 was added before filtration sterilization. Mass spectrometry analysis of samples after IdeS treatment showed a major Fc / 2 product (observed mass 26497 Da, approximately 90% of total Fc / 2), corresponding to the conjugate hRS7-3. RP-UPLC analysis of samples after IdeS treatment showed an average DAR of 3.90.

[0527] Example 6: Reduction and conjugation of hTINA to delutec

[0528] PBS (27 μL), EDTA (40 μL, 500 mM) and TCEP (153 μL, 1 mM, 2.3 equivalents) were added to the hTINA solution (1780 μL, 10 mg, 5.63 mg / mL, dissolved in PBS pH 7.4). The reaction was incubated at room temperature for 90 minutes. Subsequently, conjugation was performed by adding derutocan (200 μL, 1.8 mM, Achemblock) and incubating at room temperature for 90 minutes. The solution was then buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with PBS pH 7.4. The solution was then buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with 20 mM histidine, 6% sucrose (pH 6.0), and 0.04% Tween-20 was added before filter sterilization. RP-UPLC analysis of the reduced conjugate showed an average DAR of 3.97.

[0529] Example 7: Conjugation of hRS7(6-N3-GalNAc)2 with BCN-HS-PEG2-(eva-PAB-Ex)29 to obtain the conjugate hRS7-9

[0530] Sodium deoxycholate (4.48 mL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)2 9 (895 μL, 10 mM DMF solution) and propylene glycol (8.1 mL) were added to a solution of hRS7 (6-N3-GalNAc) 2 (22.8 mL, 448 mg, 19.64 mg / ml, dissolved in TBS pH 7.5). The reactants were incubated overnight at room temperature. Next, the conjugate was purified using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and balanced with PBS pH 7.4 on an AKTA Pure (Cytiva). In order to remove excess free payload, 100 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated for 2 h. Charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then dialyzed to 20 mM histidine, 6% sucrose buffer pH 6.0. The material was concentrated and 0.04% Tween-20 was added before filtration sterilization. Mass spectrometry analysis of samples after IdeS treatment showed a major Fc / 2 product (observed mass 26676 Da, approximately 90% of total Fc / 2), corresponding to the conjugate hRS7-9. RP-UPLC analysis of samples treated with fabricator (IdeS) showed an average DAR of 3.86.

[0531] Examples 8 to 9: In vivo studies

[0532] Example 8: In vivo efficacy study (conducted in Charles River, Morrisville, NC, USA)

[0533] NCI-N87, a human gastric cancer model cell line, was maintained in vitro, and cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0534] CB.17 SCID mice (female) aged 8-12 weeks at the start date received a subcutaneous injection in the flank region of 1×10 7 When the average tumor size reaches 150-200mm 3 At 4 pm, the mice were randomly divided into 3 groups with 8 mice in each group and the treatment was started.

[0535] Test article administration was performed via intravenous injection, and the administration volume was 10 mL / kg. Treatment started on the same day of randomization. Administration was performed in a laminar flow cabinet.

[0536] Following tumor cell inoculation, animals are examined daily for morbidity and mortality. During routine monitoring, animals are examined for tumor growth and any adverse effects of treatment on normal behavior, such as activity, visual estimates of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume is measured in two dimensions using calipers every 3-4 days, and volume data are expressed in mm3 using the formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements will be performed in a laminar flow cabinet. The experimental endpoint is a tumor volume reaching 800mm 3 , weight loss of more than 20% or 45 days (whichever comes first).

[0537] Table 1. Overview of test items and dose levels included in the in vivo studies

[0538]

[0539] exist Fig. 8A and Figure 8B Data from an efficacy study with the above test items are depicted in . Fig. 8A In vivo efficacy data over time on the NCI-N87 tumor model is shown. hRS7-3 caused tumor regression. Tumor regression was almost identical to Trodelvy, but it was given more frequently and at a higher dose level. Administration of hTINA-Drutecan caused tumor growth delay. Figure 8B Shown are changes in the body weight of mice over time.

[0540] Example 9: In vivo efficacy study

[0541] Colo-205, a colon cancer xenograft model cell line, was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with a standard 5% CO2 setting. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0542] 7-9 week old BALB / c nude mice (female) were injected subcutaneously in the right anterior flank with 5×10 6 When the average tumor size reaches 100-200mm 3At 14:00 p.m., mice were randomly divided into 5 groups of 8 mice each and treatment was started. Randomization will be based on the "matched distribution" method (Study Director TM Software, version 3.1.399.19). The date of randomization will be denoted as day 0.

[0543] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, behavioral changes were observed daily, and animals were weighed twice a week. The experimental endpoint was tumor volume reaching 3,000 mm 3 , weight loss of more than 20% or 77 days (whichever comes first).

[0544] Randomization: When the mean tumor size reached approximately 143 mm 3 A total of 40 mice were included in the NCI-H446 model study and randomly divided into 5 groups, with 8 mice in each group. TM Randomization was performed using the RT-PCR software, version 3.1.399.19. The date of randomization will be denoted as day 0. Due to the cachectic nature of the tumor model, all animals received supplemental gel from the date of randomization.

[0545] Test article administration: Test article administration was performed via intravenous injection into the tail vein, and the dosing volume was 10 mL / kg. Treatment started on the same day of randomization. Dosing was performed in a laminar flow cabinet.

[0546] Observation and data collection : After tumor cell inoculation, animals are checked daily for morbidity and mortality. During routine monitoring, animals are checked for any adverse effects of tumor growth and treatment on normal behavior, such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume is measured in two dimensions using calipers every 3-4 days, and volume data is expressed in mm3 using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Dosage and tumor and body weight measurements will be performed in a laminar flow cabinet.

[0547] Table 2. Overview of test items and dose levels included in the in vivo studies

[0548]

[0549]

[0550] Tumor volume and mouse body weight over time in the efficacy study with the above test items and the corresponding Kaplan-Meier plots are depicted in Fig.9A, 9B and 9C.

[0551] Examples 10 to 12: In vitro studies

[0552] Example 10. Human plasma stability test

[0553] The stability of ADC in human plasma was tested. Protein A agarose (1 mL agarose / mL serum) was used to deplete all IgG from plasma. ADC was added to depleted human serum at a final concentration of 0.1 mg / mL and then incubated at 37°C. At each time point, 0.5 mL was snap frozen and stored at -80°C until further analysis. To isolate ADC after incubation, 20 μl Protein A agarose resin was added to the sample and incubated at room temperature for 1 hour. The resin was washed 3 times with PBS, followed by addition of 0.1M glycine-HCl pH 2.7 (0.4mL) to elute the ADC. After elution, the sample was immediately neutralized with 1.0M Tris pH 8.0 (0.1mL). The PBS sample was spin filtered 3 times using Amicon Ultra spin filter 0.5mL MWCO 10kDa (Merck Millipore), and the volume was reduced to 40 μL, resulting in a final ADC concentration of about 1 mg / mL. Samples were analyzed by RP-UPLC (DTT reduction) on a specific date to determine the DAR, and the results are shown in the table below. At t = 7 days, the difference from t = 0 days is given as a percentage.

[0554] Table 3. Human plasma:

[0555] DAR t=0 DAR t=3 DAR t=7 hRS7-3 3.89 3.88(100％) 3.86(99％) hRS7-9 3.87 3.75(97％) 3.60(93％) hTINA-Drutecan 3.61 2.62(73％) 2.08(58％)

[0556] Example 11. Thermal stability under physiological and enhanced stress conditions

[0557] The stability of the ADC was tested at high temperature under physiological conditions (PBS, pH 7.4, 37°C) or enhanced stress conditions (citrate buffered saline, CBS, pH 5.0, 40°C). The ADC was buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1M NaOH, and balanced with PBS or CBS on an AKTA Pure (Cytiva). The solution was concentrated to a concentration of >1 mg / mL using a Vivaspin Turbo4 10kDa MWCO ultrafiltration device (Sartorius). The concentration of the ADC was measured, diluted to 1 mg / mL, and the first measurement, t=0, was performed for SE-HPLC analysis as described above. The samples were placed at 37°C or 40°C and sampled at several time points (days) and the aggregation level was determined, see Tables 4 to 6 below.

[0558] Table 4. Physiological conditions, monomer levels (percentage):

[0559] Construct t＝0 t＝1 t＝3 t＝7 hRS7-3 98.99％ 99.06％ 99.68％ 99.58％ hRS7-9 98.86％ 98.96％ 99.59％ 99.59％ hTINA-Drutecan 99.51％ 99.54％ 99.64％ 99.72％

[0560] Table 5. Physiological conditions, monomer levels (percentage):

[0561] Construct t＝0 t＝3 t＝7 hRS7-3 3.85 3.82(99％) 3.81(99％) hRS7-9 3.87 3.87(100％) 3.86(100％) hTINA-Drutecan 3.82 3.80(99％) 3.79(99％)

[0562] Table 6. Enhanced stress conditions, monomer level (percentage):

[0563] Construct t＝0 t＝1 t＝3 t＝7 hRS7-3 99.09％ 99.26％ 99.83％ 99.87％ hRS7-9 99.00％ 99.14％ 99.73％ 99.76％ hTINA-Drutecan 99.60％ 99.66％ 99.82％ 99.81％

[0564] Example 12: HIC-HPLC measurements for relative retention time determination

[0565] The samples were diluted to 1 mg / mL in PBS. Butyl-NPR HPLC column (3.5cm×4.6mm, 2.5μm) was used for HIC analysis on Agilent 1200 series. A gradient from 100% buffer A (2M ammonium sulfate in 50mM potassium phosphate pH 6.0) to 100% buffer B (50mM potassium phosphate pH 6.0+20% isopropanol) was used in 20 minutes, and 10μL samples were injected at a flow rate of 0.5mL / min. The natural mAb (hRS7 or hTINA) was measured and its retention time was set to 1. The other measured samples were relative retention times compared to the natural mAb. The results are depicted in Figure 10 and Table 7 below.

[0566] Table 7. Relative retention time of trastuzumab measured using HIC-HPLC:

[0567] Construct HIC-HPLC trace Relative retention time hTINA Figure 10(A) 1.00 hTINA-Drutecan Figure 10(B) 1.132 HkDJ Figure 10(C) 1.00 hRS7-3 Figure 10(D) 1.009 hRS7-9 Figure 10(E) 1.005

Claims

1. An antibody conjugate according to the general structure (1): AB–[(L 6 ) b –{Z–L–D} x ] y (1) in: - AB is an antibody capable of targeting tumors expressing Trop-2; -L is the linker connecting Z to D; -Z is a linking group; -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–; -D is ixotecan; -b is 0 or 1; -x is 1 or 2; and -y is 1, 2, 3, or 4.

2. The antibody conjugate of claim 1, wherein the antibody has an Fc region containing one or more mutations.

3. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody contains a V sequence having at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID No. 1 and 5. L A domain and a V domain having at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID No. 2 and 6 H domains, preferably wherein the antibody has complementarity determining regions (CDRs) having at least 90% sequence identity.

4. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is hRS7 and / or contains a light chain sequence according to SEQ ID No. 3 and a heavy chain sequence according to SEQ ID No. 4, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

5. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is hTINA and / or comprises a light chain sequence according to SEQ ID No. 7 and a heavy chain sequence according to SEQ ID No. 8, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%.

6. The antibody conjugate according to any one of claims 1 to 5, wherein the linker L has the following structure -(L 1 ) n -(L 2 ) o -(L 3 ) p -(L 4 ) q – in: -L 1 , L 2 , L 3 and L 4 Each individually is a linker, which together connect Z to D; - n, o, p and q are each independently 0 or 1, provided that n+o+p+q=1, 2, 3 or 4, preferably wherein n=o=p=1; And among them: (a) Connector L 1 It is represented by: –(W) k –(A) d –(B) e –(A) f –(B) g –(C(O)) g –, or –(W) k –(A) d –(B) e –(A) f –(C(O)) g –N*[–(A) d’ –(B) e’ –(A) f’ –(C(O)) g’ –]2, in: -d and d' are individually 0 or 1; - e and e' are independently integers ranging from 1 to 10; - f and f' are individually 0 or 1; - g and g' are independently integers ranging from 0 to 10; - k = 0 or 1, provided that if k = 1, then d = 0; -A is a sulfonamide group according to structure (23), Where a = 0 or 1, and R 13 Selected from hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The group consisting of (hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The (hetero)arylalkyl group is optionally substituted and optionally replaced by one or more selected from O, S and NR 14 The heteroatom interval, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is D linked to N via a spacer moiety, preferably wherein the spacer moiety is - (B) g –(C(O)) g –(L 2 ) o –(L 3 ) p –(L 4 ) q – -W is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–, –C(O)(CH2) m C(O)–, –C(O)(CH2) m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, wherein m is an integer ranging from 0 to 10; -B is a –CH2–CH2–O– or –O–CH2–CH2– moiety, or (B) e Yes – (CH2–CH2–O) e1 -CH2-CH2- moiety, wherein e1 is an integer in the range of 1 to 10; -N* is a branching nitrogen atom, –(A) d –(B) e –(A) f –(C(O)) g’ – Both instances are connected to it and both (C(O)) g’ Part of it is related to –(L 2 ) o –(L 3 ) p –(L 4 ) q –D connection, where L 2 , L 3 , L 4 , o, p, q and D are each selected individually; and / or (b) Connector L 2 is a peptide spacer, preferably comprising 1 to 5 amino acids, more preferably a dipeptide, tripeptide or tetrapeptide spacer, most preferably wherein L 2 Represented by the general structure (L3): Where R 17 =CH3 or CH2CH2CH2NHC(O)NH2; and / or (c) Connector L 3 is a self-immolative spacer, preferably a p-aminobenzyloxycarbonyl (PABC) derivative according to structure (L4), Where R 21 It is H, R 26 or C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl groups, C3–C 10 Alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally replaced by one or more selected from O, S and NR 28 The heteroatom interval, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, Preferably, R 21 is H or C(O)R 26 , where R 26 =4-methyl-piperazine or morpholine, most preferably wherein R 21 It is H; and / or (d) Connector L 4 According to the structure – NR 22 –(C z -alkylene)-C(O)-aminoalkanoic acid spacer, wherein x is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl; or Connector L 4 According to the structure – NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -C(O)-, wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R 22 is H or C1–C4 alkyl; or Connector L 4 According to the structure – NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer, wherein h is 0 or 1, x is an integer in the range of 1 to 10 and R 22 is H or C1–C4 alkyl.

7. The antibody conjugate according to any one of claims 1 to 6, wherein b=1 and the conjugation is via the glycan of the antibody, preferably wherein j=0.

8. The antibody conjugate according to any one of claims 1 to 7, wherein Z-L-D has a structure selected from the group consisting of (X) to (XII): The wavy line indicates the connection to Z, and L 2 , L 4 , o and q as defined in claim 6, Preferably wherein Z-LD has a structure selected from the group consisting of (Xa), (XIb), (XIIg), (XIIIe) and (XIIh): The wavy line indicates the connection to Z, and R 22 As defined in claim 6.

9. The antibody conjugate according to claim 8, which is according to structure (XII) or (XIII) and wherein o=1 and the antibody is hRS7 as defined in claim 5 or hTINA as defined in claim 6, preferably wherein q=0.

10. The antibody conjugate according to claim 10, which is according to structure (XII) or (XIII), wherein q=0, o=1, L 2 is a peptide spacer containing 1 to 5 amino acids, the antibody is hRS7 as defined in claim 5, and the payload is exotecan.

11. The antibody conjugate according to claim 1, which is according to structure (XIIIe) as defined in claim 8, and wherein the antibody is hRS7 as defined in claim 5.

12. A method for preparing an antibody conjugate according to any one of claims 1 to 11, comprising: (i) combining an antibody comprising a y-core N-acetylglucosamine (GlcNAc) portion with a compound of formula S(F) x -P compound in the presence of a catalyst, wherein y = 1, 2, 3 or 4, wherein S (F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The core-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26): AB–[GlcNAc(Fuc) w –S{F} x ] y (26) in - AB is an antibody capable of targeting tumors expressing Trop-2; -Fuc is fucose; -w is 0 or 1; and (ii) reacting the modified antibody with a compound according to structure (2): Q–L–D (2) in: -Q is the reactive moiety; -L is the linker connecting Z to D; -D is ixotecan; To obtain the antibody conjugate according to structure (1).

13. The process according to claim 12, wherein the reaction of step (ii) is a nucleophilic reaction or a cycloaddition, preferably a 1,3-dipolar cycloaddition, and preferably wherein Q comprises or is an alkyne moiety and F comprises or is an azide moiety.

14. The method according to claim 12 or 13, wherein Q is a click probe comprising a (hetero)cycloalkyne moiety or a (hetero)cycloalkene moiety, preferably wherein the click probe Q is selected from (Q21) to (Q38) and (Q44) to (Q56): in -B is an anion; and - (Q50) and (Q51) wherein the R group on Si is an alkyl group or an aryl group; And wherein F is a click probe selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene and Sydney ketone, preferably F is an azide moiety.

15. A method for targeting a cell expressing Trop-2, comprising contacting the antibody conjugate according to any one of claims 1 to 11 with a cell that may express Trop-2, preferably wherein the cell is a tumor cell expressing Trop-2.

16. The method of claim 15, wherein the targeting of Trop-2-expressing cells comprises one or more of the following: treating, imaging, diagnosing, preventing proliferation of, inhibiting and reducing Trop-2-expressing cells, particularly Trop-2-expressing tumor cells.

17. The method of claim 15 or 16, wherein the subject has oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer, or lung cancer.

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