Antibody conjugates for targeting Trop-2-expressing tumors

Abstract

The present invention relates to an antibody conjugate that is particularly suitable for targeting Trop-2 expressing cells, especially tumor cells. The antibody conjugate according to the invention has the structure (1), where AB is an antibody capable of targeting Trop-2 expressing tumors, L is a linker connecting Z to D, Z is a connecting group, and L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which 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, 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 exatecan, b is 0 or 1, x is 1 or 2, and y is 1, 2, 3, or 4. The present invention further relates to methods for preparing the antibody conjugates of structure (1) and applications of the antibody conjugates of structure (1).

Description

[Technical field]

[0001] The present invention is in the field of bioconjugation. More particularly, the present invention relates to antibody-drug conjugates for targeted treatment of patients with cancer, particularly Trop-2 expressing tumors. [Background technology]

[0002] A promising approach for targeted treatment of tumors involves conjugating multiple (2-8) highly toxic payloads to monoclonal antibodies, thereby generating antibody-drug conjugates (ADCs). ADCs are well known in the art, for example, as described by Chari et al., Angew. Chem. Int. Ed. 2014, 53, 3796, and Beck et al., Nat. Rev. Drug Discov. 2017, 16, 315-37. Mechanistically, antibodies are designed to bind with high specificity to tumor-associated receptors that are overexpressed compared to healthy tissues. ADCs are believed to internalize into tumor cells after binding to the receptor, and then release the toxic payload upon degradation of the antibody and / or linker in the lysosome.

[0003] Current ADCs are commonly prepared by a variety of conjugation techniques (summarized in Figure 1), primarily based on conjugation to cysteine ​​side chains bearing maleimides or lysine side chains bearing activated esters. To generate ADCs based on native cysteines that naturally participate in disulfide bonds, the thiols in the side chains can be liberated by exposure of the antibody to a suitable reducing agent, such as TCEP or DTT, followed by treatment with a maleimide-functionalized linker drug. The resulting ADCs typically consist of a mixture of positional isomers if only the total of the eight free thiols are not comprehensively alkylated. Alternatively, to generate site-specific ADCs, antibodies can be generated by mutating one or more amino acids to cysteines at defined positions in the sequence, whose side chains can be selectively liberated 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, such as HC-i239C, can also be inserted into the sequence. In addition to maleimides as alkylating agents, reactions of cysteine ​​side chains with haloacetamides or vinylbenzene derivatives have also been reported. In addition to reacting with natural amino acid side chains, certain unnatural (non-standard) amino acids can also be engineered into the amino acid sequence of an antibody, thereby providing unique handles for chemical conjugation such as ketones, acetylenes, azides, cyclic alkynes or cyclic alkenes for reaction with oximes, azides, alkynes or tetrazines, respectively. However, the drawback of the latter approach is that the native sequence of the antibody must be redesigned, which, in addition to being time-consuming and expensive, can lead to instability problems.

[0004] Glycan-mediated conjugation via oxidative ligation sequences is known in the art and has been described, for example, by Hamann et al. (Bioconjugate Chem. 2002, 13, 47-58). Glycan-mediated chemoenzymatic conjugation is known in the art and has been described, for example, by Boons et al., Angew. Chem. Int. Ed. 2014, 53, 7179 for the use of sialyltransferases, and by Zhu et al., mAbs 2014, 6, 1 and Cook et al., Bioconjugate Chem. 2016, 27, 1789 for the use of mutant galactosyltransferases.

[0005] Glycan-mediated chemoenzymatic conjugation, including initial trimming of the glycan, is known in the art and described by van Geel et al, Bioconjugate Chem. 2015, 26, 2233, and is shown diagrammatically in FIG. 2. Briefly, monoclonal antibodies are treated with endoglycosidases to trim the glycan to the core GlcNAc (directly attached to Asn-297), followed by the transfer of the azido-modified sugar under the action of a glycosyltransferase. Various structures of UDP-azido sugars are shown in FIG. 3. One particularly suitable combination involves the transfer of GalNAz 2b (2-azidoacetyl-N-galactosamine) under the action of the mutant galactosyltransferase GalT (Y289L) disclosed in WO2007 / 095506, EP2911699B1, and van Geel et al. An alternative potent combination involves 6-azidoGalNAc 2d with a native GalNAc transferase, as disclosed in PCT / EP2016 / 059194. Another useful combination involves GlcNAz with α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT1), as disclosed in WO2021 / 248048.

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

[0007] The payload of ADCs is typically in the low nanomolar or picomolar range with IC 50 The most suitable cytotoxic molecules for ADCs are highly cytotoxic molecules, particularly low to medium molecular weight compounds (e.g., about 200 to about 2500 Da). Examples of suitable cytotoxin classes for ADCs include anthracyclines, camptothecins, taxanes, tubulysins, enediynes, inhibitory peptides, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, hemiasterlin, BCL-XL inhibitors, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof. A representative set of cytotoxic molecules, and / or synthetic derivatives or prodrugs thereof, with suitable attachment points for conjugation to monoclonal antibodies is shown in FIG. 5.

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

[0009] Specific examples of camptothecins suitable for ADC applications include, but are not limited to, SN-38, exatecan, exatecan-S, topotecan, ciratecan, cositecan, lurtotecan, gimatecan, belotecan, rubitecan, AMDCPT, G-AMDCPT, and other synthetic camptothecins whose structures are shown in Figure 6. Various novel camptothecins have been disclosed in EP0296597, WO2019 / 236954, WO2020 / 200880, WO2020 / 219287, CN113816969, CN113710277, and US20180200273.

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

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

[0012] Proteins with roles in breast cancer proliferation, differentiation, invasion and / or metastasis can affect the biological progression of tumors and therefore provide important prognostic information. One such candidate is Trop-2 (GA733-1, EGP-1), a 45 kDa monomeric transmembrane glycoprotein belonging to the TACSTD gene family, specifically TACSTD2, which is expressed in human epithelial cells at diverse stages of differentiation. Overexpression of Trop-2 has been shown to be necessary and sufficient to stimulate tumor growth and is associated with poor overall prognosis. Expression of Trop-2 has been associated with poor prognosis in several human cancers, including oral, pancreatic, gastric, ovarian, colorectal, breast and lung tumors. For example, Trop-2 overexpression was observed in 55% of pancreatic cancer patients studied, positively correlated with metastasis, tumor grade and poor progression-free survival in patients undergoing surgery with curative intent. Similarly, in gastric cancer, 56% of patients could demonstrate Trop-2 overexpression on their tumors, which also correlated with shorter disease-free survival and poor prognosis in patients with lymph node involvement of Trop-2-positive tumor cells.

[0013] Considering these features, and the fact that Trop-2 is associated with a great many intractable cancers, it is an attractive target for therapeutic intervention. Nevertheless, it must be considered that Trop-2 is also expressed in some normal tissues, but usually at a much lower intensity compared to that in tumor tissues, and often in areas of tissues with limited vascular access.

[0014] Several monoclonal antibodies against Trop-2 have been established. Several anti-Trop-2 monoclonal antibodies, such as 77220, are commercially available as reagents. Some of these established anti-Trop-2 monoclonal antibodies are under investigation for cancer therapy. Most of the currently commercially available anti-Trop-2 monoclonal antibodies, such as T16, were generated using myeloma cell lines such as NS-1 or SP2-1 as fusion partners that retained the expression of the parent immunoglobulin light chain. This resulted in these antibodies being in fact a heterogeneous mixture of antibodies with one, both, or neither of the light chains directly involved in Trop-2 recognition.

[0015] WO1997 / 14796 describes a monoclonal antibody BR110 that is known to bind to Trop-2 on the cell surface and internalize into the cell. Patent applications WO2003 / 074566, US2004 / 001825, US2007 / 212350 and US2008 / 131363, and patents US10,179,171B2 and EP3483183B1 teach RS7 antibodies and their use in tumor therapy and diagnosis. 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 therapy. Anti-Trop-2 monoclonal antibody AR47A6.4.2 is disclosed in WO2007 / 095748, and AR52A301.5 is disclosed in WO2007 / 095749, both of which are antibodies that specifically target Trop-2 expressing cancer cells to be effective. Patent application WO2008 / 144891 teaches a humanized version of AR47A6.4.2 as an anti-Trop-2 monoclonal antibody for the treatment of tumors. Patent application WO2011 / 155579 (Sapporo) teaches the humanized monoclonal antibody AR47A6.4.2 or its antibody fragments that bind with high affinity to the extracellular domain of human Trop-2 and exhibit high ADCC activity and high anti-tumor activity. Patent applications WO2013 / 077458 and US9427464B2 (LivTech / Chiome) teach anti-human Trop-2 antibodies with anti-tumor activity, particularly humanized antibodies including Huk5-70-2, particularly anti-human Trop-2 antibodies with anti-tumor activity in vivo. Patent applications WO2013 / 068946 and US8,871,908B2 (Rinat / Pfizer) teach the antibody 7E6 and humanized h7E6 that specifically bind to trophoblast cell surface antigen-2 (Trop-2). The invention further relates to therapeutic methods for using these antibody conjugates for the treatment of conditions associated with Trop-2 expression (e.g. cancer), such as colon, esophageal, gastric, head and neck, lung, ovarian, or pancreatic cancer. Patent US8,715,662 (Oncoxx) teaches the antibody 2G10 that specifically binds 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 / sacituzumab and conjugated to the camptothecin analog SN-38 via an acid-cleavable linker CL2A as disclosed in WO2015 / 012904, and is currently in late-stage 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 and conjugated to the camptothecin analog DXd via a protease-sensitive GGFG-cleavable linker as disclosed in WO2015 / 098099, US11,008,398B2, and EP3088419B1, and is in clinical evaluation for the treatment of solid tumors. Third, PF-06664178 is an ADC derived from the monoclonal antibody RN926 site-specifically conjugated to the auristatin analog PF-06380101 under the action of microbial transglutaminase. PF-06664178 was evaluated in a Phase I clinical trial in patients with advanced or metastatic solid tumors, but the ADC was discontinued due to toxicity at high dose levels and limited antitumor activity. Summary of the Invention [Problem to be solved by the invention]

[0017] The inventors have developed antibody conjugates which are highly suitable for targeting Trop-2 expressing cells, in particular tumors, and are therefore highly suitable for the treatment of Trop-2 positive cancers, in particular breast, colorectal, pancreatic, lung, bladder, head and neck, ovarian or esophageal cancer. [Means for solving the problem]

[0018] In a first aspect, the invention relates to an antibody conjugate. In this regard, in a second aspect, the invention relates to a process for preparing an antibody conjugate according to the invention. In a third aspect, the invention relates to a method for targeting Trop-2 expressing cells. In this regard, a first medical use of an antibody conjugate according to the invention as well as a second medical use for the treatment of cancer. In a last aspect, the invention relates to the use of a conjugation mode to increase the therapeutic index of an antibody conjugate in the treatment of Trop-2 expressing tumors.

[0019] definition As used in this description and claims, the verb "comprise" and its conjugations are used in its open-ended sense, meaning that items following the word are included but not excluding items not specifically mentioned.

[0020] In addition, reference to an element by 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 only one of the element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0021] A linker is defined herein as a moiety that connects (covalently bonds) two or more elements of a compound. A linker may include one or more spacer moieties. A spacer moiety is defined herein as a moiety that separates (i.e., provides distance between) two (or more) parts of a linker and covalently bonds them to each other. A linker can be, for example, part of a linker construct, a linker conjugate, a linker payload (e.g., a linker drug) or an antibody conjugate, as defined below.

[0022] "Hydrophilic group" or "polar linker" is defined herein as any molecular structure that contains one or more polar functional groups that impart improved polarity and therefore improved water solubility to the molecule to which it is attached. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonic acid groups, phosphate groups, acylsulfamide groups or carbamoylsulfamide groups. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency and improved pharmacokinetics that result in less aggregation, higher efficacy and in vivo tolerability when incorporated into antibody-drug conjugates.

[0023] The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced with a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharma- ceutically acceptable salt, although this is not necessary for salts that are not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation with the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term "pharma-ceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts may be derived from pharma-ceutically acceptable inorganic or organic bases and pharma-ceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharma- ceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0024] The term "enediyne" or "enediyne antibiotic" or "enediyne-containing cytotoxin" refers to any cytotoxin characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule known in the art, including neocarzinostatin (NCS), C-1027, kedarcidin (KED), madulopeptin (MDP), N1999A2, sporolide (SPO), cyanosporacids (CYA and CYN), as well as physiolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamemycin, shishijimicin, and unicaramycin (UCM).

[0025] The term "alkylaminosugar," as used herein, refers to a tetrahydropyranyl moiety connected through its 2-position to an alcohol functional group, thereby forming an acetal functional group, and further substituted with (at least) one N-alkylamino group at positions 3, 4 or 5. An "N-alkylamino group" in this context refers to an amino group bearing one methyl, ethyl, or 2-propyl group.

[0026] The term "click probe" refers to a functional moiety capable of undergoing a click reaction, i.e., two compatible click probes click 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, a click probe Q in a compound according to the invention can react with a click probe F on a (modified) protein such that, upon the occurrence of a click reaction, 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.

[0027] An "acylsulfamide moiety" is defined herein as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamoylated at one end of the molecule and N-alkylated (mono- or bis) 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."

[0028] A "domain" is generally defined on the basis of sequence homology and can be any region of a protein that is often associated with a particular structural or functional entity. The term domain is used herein to designate either an individual Ig-like domain, such as a "V domain," or a group of contiguous domains, such as a "C2 type 1-2 domain."

[0029] A "coding sequence," or a sequence that "encodes" an expression product, e.g., an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, upon expression, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0030] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as, for example, promoter sequences, which determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may cover genomic sequences that code for proteins, i.e., sequences that include regulatory elements, promoters, introns, and exon sequences.

[0031] The term "glycoprotein" is used herein in its ordinary scientific sense to refer to a protein that contains one or more mono- or oligosaccharide chains ("glycans") covalently attached to the protein. The glycans may be attached to a hydroxyl group on the protein (O-linked glycans), e.g., the hydroxyl group of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline, or to an amide functional group on the protein (N-glycoproteins), e.g., asparagine or arginine, or to a carbon on the protein (C-glycoproteins), e.g., tryptophan. A glycoprotein may contain more than one glycan, may contain a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may contain a combination of N-linked glycans, O-linked glycans, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and thus qualify as glycoproteins. Examples of glycoproteins include PSMA (prostate specific membrane antigen), CAL (Candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins and antibodies.

[0032] The term "glycan" is used herein in its normal scientific sense to refer to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term glycan refers to the carbohydrate moiety of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of one sugar, which may be without further substitution (monosaccharide) or may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when a longer glycan chain is linked to a protein, the glycan is also considered herein as a glycan. The glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar that is directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but is attached to a single other sugar, i.e., has no further sugar substituents at one or more of the other hydroxyl groups, is called a terminal sugar. For the avoidance of doubt, there may be multiple terminal sugars in the oligosaccharide of a glycoprotein, but only one core sugar. A glycan may be an O-linked glycan, an N-linked glycan, or a C-linked glycan. In an O-linked glycan, the monosaccharide or oligosaccharide glycan is attached to an O atom in an amino acid of the protein, typically through a hydroxyl group of serine (Ser) or threonine (Thr). In an N-linked glycan, the monosaccharide or oligosaccharide glycan is attached to a protein through an N atom in an amino acid of the protein, typically through the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In a C-linked glycan, the monosaccharide or oligosaccharide glycan is attached to a C atom in an amino acid of the protein, typically the C atom of tryptophan (Trp).

[0033] The term "antibody" (AB) is used herein in its normal scientific sense. An antibody is a protein produced by the immune system that is capable of recognizing and binding to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein 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 herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" includes whole antibodies, but is also intended to include antibody fragments, such as truncated antibodies, scFv-Fc fragments, minibodies, diabodies, or antibody Fab fragments, F(ab')2, Fv fragments, or Fc fragments derived from scFv. Furthermore, 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.

[0034] An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). Light chains contain two domains or regions: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to antigens. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g., human G1m1, G1m2, Gm3, 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). 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. Antibodies can be engineered in the Fc domain to enhance or abolish binding to Fc-gamma receptors, 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 Fcγ receptors can also be achieved by mutating the N297 amino acid to any other amino acid except asparagine, mutating the T299 amino acid to any other amino acid except threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies, for example, with PNGase F or endoglycosidase. Immunoglobulins can be from any species, including from human, mouse, or rabbit. Each chain contains a different sequence domain.

[0035] The percentage of "sequence identity" may be determined by comparing two optimally aligned sequences over a comparison window, where portions of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the entire length of the reference sequence.

[0036] The term "CDR" refers to complementarity determining region, and the specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. An antibody binding site is composed of residues that are primarily from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) also influence the overall domain structure and thus the binding site. Thus, complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin have three CDRs, CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0037] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody molecule of a single amino acid sequence directed against a particular antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of a B cell or hybridoma, but may also be recombinant, i.e., produced by protein engineering.

[0038] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises the VH and VL domains of an antibody from a non-human animal associated with the CH and CL domains of another antibody, in one embodiment a human antibody. As a non-human animal, any animal can be used, such as mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also represent multispecific antibodies, having specificity for at least two different antigens.

[0039] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified to avoid or minimize immune responses in humans, for example by replacing certain amino acids in the framework regions of the VH and VL domains. The constant domains of a humanized antibody are most often human CH and CL domains. A "fragment" of a (conventional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a heavy chain antibody or a single domain antibody such as a VHH. [Brief description of the drawings]

[0040] [Figure 1]Shown below is a representative set of reactive groups (F) that, when present in a biomolecule, yield a connecting group Z (1a-1h) upon reaction with reactive group Q. The functional group F can be artificially introduced (engineered) into the biomolecule at any position of interest. [Diagram 2] It is shown diagrammatically how an antibody conjugate can be obtained from any monoclonal antibody in a two-step process. In the first step, an azide-modified UPD-Gal or UDP-GalNAc can be conjugated to the monoclonal antibody in a one-pot process involving (a) trimming of the glycan (to the core GlcNAc) by an endoglycosidase and (b) attachment of the azido sugar under the action of a glycosyltransferase (galactosyltransferase or a mutant thereof or GalNAc transferase), thereby generating a β-glycosidic 1-4 linkage between the azide-modified GalNAc and the GlcNAc. In the second step, the azide-modified antibody can be reacted with an appropriately functionalized cyclooctyne, thereby generating the antibody conjugate. [Diagram 3] For example, structures of some derivatives of the UDP-sugar of galactosamine are shown that can be modified with a 3-mercaptopropionyl group (2a), an azidoacetyl group (2b), or an azidodifluoroacetyl group (2c) at the 2-position of N-acetylgalactosamine, or with an azido group (2d) at the 6-position. [Figure 4] Preferred choices for reactive moiety Q are cyclooctynes ​​(A-T) suitable for metal-free click chemistry. [Diagram 5] 5 shows a set of exemplary toxic payloads for conjugation to various Trop-2 targeting monoclonal antibodies according to the present invention. The attachment point of the linker (to the amino group present in the payload) is indicated by an arrow. A preferred conjugate according to the present invention contains a payload that includes an attachment point as shown in FIG. [Figure 6] 1 shows the structures of various camptothecins, which are preferred payloads in the context of the present invention. [Figure 7]1 shows the structures of BCN-linker drugs containing Val-Cit-PABC or Val-Ala-PABC cleavable linkers used to prepare ADCs via click chemistry to azido-sugar remodeled antibodies with the following payloads (3=exatecan, 4=MMAE, 5a=calicheamicin γ1 I, 5b=glycine-calicheamicin γ1 I, 6=6-aminohexanoyl-maytansinoid). Particularly preferred is linker-drug construct 3. [Figure 8] Figure 8 shows in vivo data in N87 mouse xenograft model of sacituzumab-3 (DAR4) versus DS-1062, a Phase 3 clinical ADC (DAR4) consisting of datopotamab / hTINA antibody and deruxtecan linker-payload (based on GGFG cleavable linker and DXd payload). After administration of a single dose of 10 mg / kg (respectively), substantially better tumor regression was observed with sacituzumab-3, followed by datopotamab-deruxtecan after 18 days (Figure 8(A)) or 31 days (Figure 8(C)). Figures 8(B) and 8(D) show body weight plots up to 18 or 31 days. [Figure 9] Shows in vivo efficacy data in Colo-205 mouse xenograft model. hTINA-deruxtecan at both dose levels shows low antitumor efficacy, while hRS7-3 shows significant antitumor efficacy. Even at the dose level of 8mg / kg, a clear dose response is observed, inducing complete tumor regression from day 25 to the end of study day 70. [Figure 9B] The body weight of the mice over time is shown, and a Kaplan-Meier plot is shown in Figure 9C. [Figure 10] 1 shows the HIC-HPLC trace of Example 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] In a first aspect, the present invention relates to an antibody-conjugate of general structure (1): AB-[(L 6 ) b -{ZLD} x ]y (1) Where: -AB is an antibody capable of targeting Trop-2-expressing tumors, - L is a linker connecting Z to D, -Z is a connecting group; -L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which 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, 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 selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; -b is 0 or 1, -x is 1 or 2, -y is 1, 2, 3, or 4.

[0042] Salts, preferably pharma-ceutically acceptable salts, of the antibody conjugates according to structure (1) are also contemplated in the present invention.

[0043] In a second aspect, the present invention relates to a process for preparing an antibody conjugate according to the present invention, comprising reacting a compound according to general structure (2) with an antibody (3). The compound according to general structure (2) comprises a reactive moiety Q and an antibody-reactive moiety F capable of reacting with Q in a conjugation reaction, where Q and F react to form a linking group Z. In this reaction, a conjugate according to general structure (1) is formed. The process according to this aspect relates to the following bioconjugation reaction: AB-[(L 6 ) b -{F} x ] y +ZLD→AB-[(L 6 ) b -{ZLD} x ] y (3) (2) (1)

[0044] In the following, an antibody conjugate according to structure (1) is first defined. The structural features of an antibody conjugate according to structure (1) also apply to a compound according to structure (2) and an antibody according to structure (3), since they are invariant in the conjugation reaction, except for the reactive moieties F and Q, which are converted to a linking group Z upon reaction of a compound according to structure (2) with an antibody according to structure (3).

[0045] In a third aspect, the present invention relates to an application antibody conjugate according to structure (1) for targeting Trop-2 expressing cells. In this regard, the present invention relates to a first medical use and a second medical use of an antibody conjugate according to structure (1).

[0046] As will be appreciated by one of skill in the art, the definitions of chemical moieties, and their preferred embodiments, apply to all aspects of the present invention.

[0047] Antibody conjugates of general structure (1) In a first aspect, the present invention relates to an antibody-conjugate of general structure (1): AB-[(L 6 )b -{ZLD} x ] y (1) Where: -AB is an antibody capable of targeting Trop-2-expressing tumors, -b is 0 or 1, -L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which 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, 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-, -Z is a connecting group; - L is a linker connecting Z to D, D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; -x is 1 or 2, -y is 1, 2, 3, or 4.

[0048] Antibody AB The antibody conjugate according to the invention contains an antibody capable of targeting Trop-2 expressing cells, especially tumor cells. Trop-2 is a known target for cancer therapy. The term "expressing" is commonly used in the art and refers to the overexpression of a target relative to its expression in healthy tissue. Antibodies capable of targeting Trop-2 expressing tumors are also referred to as anti-Trop-2 antibodies, Trop-2 targeting antibodies, or Trop-2 binding antibodies. Anti-Trop-2 antibodies selectively bind to Trop-2 expressing cells.

[0049] Anti-Trop-2 antibodies are known in the art and any suitable one can 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.

[0050] Here, the Fc regions of these antibodies may have one or more mutations, such as 0-10 mutations or 0-5 mutations. Mutations that alter binding to the FcRn receptor to modulate the half-life of the antibody are particularly preferred. For example, inclusion of the mutations Met252Tyr, Ser254Thr, and Thr256Glu, often referred to as YTE, in an IgG1 Fc results in approximately 11-fold higher binding of the antibody to human FcRn, thereby increasing the circulating half-life by approximately 3.5-fold. Thus, in one embodiment, the antibody has a circulating half-life of 2.5×10 -6 Less than M, preferably 0.05 to 0.99 × 10 -6 M, more preferably 0.1 to 0.49 × 10 -6 M, most preferably 0.2 to 0.4 × 10 -6 Apparent human FcRn binding affinity K in the range of M D,app The apparent binding affinity K D,app can be determined according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, the antibody AB is a YTE variant of a preferred antibody as defined above or below.

[0051] hRS7, also known as sacituzumab, may be defined as comprising 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, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0052] hTINA, also known as datopotamab, may be defined as comprising a light chain sequence according to SEQ ID NO: 7 and a heavy chain sequence according to SEQ ID NO: 8, with sequence identity of 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, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0053] Alternatively, the antibody may be L Domain and V H In a preferred embodiment, the antibody is defined by a variable domain, which together form an antigen-binding variable domain. Thus, in a preferred embodiment, the antibody comprises a variable domain selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 19, 21 and 23. L domain and a V selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 15, 20, 22 and 24 HIn a further preferred embodiment, the antibody comprises the V domain of SEQ ID NO: 1 or 5, which has a sequence identity of at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, and most preferably at least 99% or 100%. L Domain and V of SEQ ID NO: 2 or 6 H In a particularly preferred embodiment, the antibody comprises the V domain of SEQ ID NO:1, with sequence identity of at least 70%, preferably at least 75%, or at least 80%, more preferably at least 85% or at least 90% or at least 95%, and most preferably at least 99% or even 100%. L Domain and V of SEQ ID NO:2 H domains, the sequence identity being at least 70%, preferably at least 75%, or at least 80%, more preferably at least 85% or at least 90% or at least 95%, and most preferably at least 99% or even 100%.

[0054] The sequence identity described above is L Domain and V H The term "V" refers to the complete sequence of the domain. The overall sequence of these domains allows for some variation in the sequence without compromising binding to Trop-2, however, it is preferred that the complementarity determining regions (CDRs) have a higher sequence identity to ensure that binding to Trop-2 is not significantly compromised. The positions of the CDRs are shown in the table below. Thus, the antibody may comprise a V or VD polypeptide selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 19, 21, and 23. L domain and a V selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 15, 20, 22, and 24 H and preferably the V domain of SEQ ID NO: 1 or 5 L Domain and V of SEQ ID NO: 2 or 6 H and more preferably the V domain of SEQ ID NO:1 L Domain and V of SEQ ID NO:2 HPreferably, the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. L Domain and V H It will be appreciated that the domains may be combined with appropriate constant domains to form a complete antibody.

number

number

[0055] In a particularly preferred embodiment, the antibody comprises the V 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 In the present specification, the sequence identities as defined above for the complete sequence and for the CDRs apply. Most preferably, the antibody comprises the V domain of SEQ ID NO: 1. L Domain and V of SEQ ID NO:2 H The domain.

[0056] Alternatively, an antibody is defined by its light and heavy chains that together form the antibody. Thus, in a preferred embodiment, the antibody comprises 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, with a sequence identity of 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 comprises 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, with a sequence identity of 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 the most preferred embodiment, the antibody comprises 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%, and most preferably at least 99% or even 100%.

[0057] The aforementioned sequence identities refer to the complete sequences of the light and heavy chains. The entire sequences of these chains allow some variations in the sequence without compromising the binding to Trop-2, but it is preferred that the CDRs have a higher sequence identity to ensure that the binding to Trop-2 is not significantly compromised. The positions of the CDRs are shown in the table below. Thus, it is preferred that the antibody comprises a light chain selected from the group consisting of SEQ ID NOs: 3, 7, 11 and 16 and a heavy chain selected from the group consisting of SEQ ID NOs: 4, 8, 12, 17 and 18, with the sequence identity of the CDRs being at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%.

number

number

[0058] In a particularly preferred embodiment, the antibody comprises 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, where the sequence identities defined above for the complete sequence and for the CDRs apply. Particularly preferred is this antibody with exatecan as payload D.

[0059] Linker L 6 When the reactive group F is directly attached to the antibody, or even to a part of the antibody structure, a linker L connecting AB to F (in the case of an antibody of structure (3)) or AB to Z (in the case of a conjugate of structure (1)) can be used. 6 is absent and b=0. This is the case, for example, for cysteine ​​and lysine conjugations. Alternatively, the linker L connecting AB to F (in the case of an antibody of structure (3)) or AB to Z (in the case of a conjugate of structure (1)) 6 A reactive group F can also be introduced into the antibody using 6 exists and b=1. L 6 If present, the reactive group F is typically introduced into the glycan of the antibody. This is the case, for example, for conjugation via an artificially introduced reactive group F, such as, for example, using transglutaminase or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase). For example, the modified sugar residue S(F) xmay be introduced into the glycan to extend the glycan with one monosaccharide residue S that introduces x reactive groups F onto the glycan of the antibody. In the most preferred embodiment, conjugation occurs via the glycan of the antibody, and b=1. The site of conjugation is preferably on the heavy chain of the antibody.

[0060] If present, L 6 is a linker connecting AB to F or Z, -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which 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, 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-. Typically, L 6 is formed at least in part by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at the asparagine residue at or near position 297 of the heavy chain, which is modified by complex-type glycans. This naturally occurring glycosylation site of the antibody is preferably used, although other glycosylation sites, including artificially introduced ones, may also be used in the linker L. 6 Therefore, in a preferred embodiment, L 6 is attached to an amino acid of the antibody located at a position within 250 to 350 of the heavy chain, preferably within 280 to 310 of the heavy chain, more preferably within 295 to 300 of the heavy chain, and most preferably at position 297 of the heavy chain.

[0061] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan of an antibody, or a part thereof. Thus, the -GlcNAc(Fuc) of the glycan w -(G) j- is typically derived from the original antibody, GlcNAc is an N-acetylglucosamine moiety, and Fuc is a fucose moiety. Fuc is typically attached to GlcNAc via an α-1,6-glycosidic bond. Usually, the antibody can be fucosylated (w=1) or non-fucosylated (w=0). 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. The GlcNAc residue, which may also be referred to as the core-GlcNAc residue, is the monosaccharide that is directly attached to the peptide portion of the antibody.

[0062] S is core-GlcNAc(Fuc) w The moiety may be directly attached to the core-GlcNAc(Fuc) moiety, i.e., j=0, which means that the remainder of the glycan is connected to the core-GlcNAc(Fuc) moiety before S is attached. w Such trimming of glycans is well known in the art and can be accomplished by the action of endoglycosidases. Alternatively, the core-GlcNAc(Fuc) w There is one or more monosaccharide residues between the 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) j is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer ranging from 2 to 5. j is typically linked to GlcNAc(Fuc) via a β-1,4 bond. wIn a preferred embodiment, j is 3, 4 or 5. Any monosaccharide that may be present in a glycan may be used as G, but each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and N-acetylneuraminic acid. More preferred choices for G are galactose, N-acetylglucosamine, and mannose. The inventors have found that antibody conjugates with j less than 4 exhibit no or little binding to Fc-gamma receptors, while antibody conjugates with j in the range of 4 to 10 bind to Fc-gamma receptors. Thus, by selecting a particular value of j, a desired degree of binding to Fc-gamma receptors can be obtained. Thus, it is preferred that 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.

[0063] S is a sugar or sugar derivative. The term "sugar derivative" is used herein to indicate a monosaccharide sugar, i.e. a derivative of a monosaccharide sugar, including a substituent and / or a functional group. Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), 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.

[0064] x is an integer indicating the number of linking groups Z (of conjugate (1)) or reactive groups F (of antibody (3)) attached to the sugar (derivative) S. Thus, an 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 attached to a single occurrence of S. x is 1 or 2, preferably x=1.

[0065] The linking group Z (in the case of conjugate (1)) or the reactive group F (in the case of antibody (3)) may be directly attached to S or may be linked by a linker L present between S and Z or F. 7 may be present. 7 is a linker connecting S to Z. 7 may be present (w'=1 or 2) or absent (w'=0). Typically, each moiety Z is linked to a linker L 7 and thus in one embodiment, w′=0 / x. 7 is absent and each binding site Z is directly attached to S. If present, L 7 may be selected from -N(H)C(O)CH-, -N(H)C(O)CF- or -CH-. In a preferred embodiment, x=1 and w'=0 or 1, most preferably x=1 and w'=0.

[0066] y is an integer representing the number of sugar(s) (derivative(s)) S, each of which has x reactive groups F or is connected to x connecting groups Z, which are connected to the antibody. y is 1, 2, 3, or 4, preferably y=2 or 4, and most preferably y=2. Thus, the antibody comprises y moieties S, each of which comprises x reactive moieties F. Each of these reactive moieties F reacts with a reactive moiety Q of a compound according to general structure (2), resulting in the formation of x+y connecting groups Z, and x+y compounds according to general structure (2) being attached to a single antibody. Each compound according to general structure (2) can be, for example, a nitrogen atom N in L. *By branching of D, multiple payloads can be included. Each compound according to general structure (2) preferably contains one or two occurrences of D, most preferably two occurrences of D. In a particularly preferred embodiment, the linker L 1 The second occurrence of D is connected to a branched nitrogen atom, N * Contains:

[0067] The amount of payload (D) molecules bound to a single antibody is known in the art as DAR (drug-antibody ratio). In the context of the present invention, DAR is preferably an integer in the range of 1 to 8, more preferably 2 or 4, most preferably DAR=4. Alternatively, DAR is preferably an integer in the range of (x+y) to [(x+y)×2], most preferably DAR=[(x+y)×2]. For preferred values ​​of x of 1 and y of 2, DAR is preferably 4. It will be understood that these are theoretical DAR values, and in practice, the DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as stochastic mixtures of antibody-drug conjugates, DAR values ​​vary between individual conjugates, and depending on the conjugation technique used, the DAR may have a wide distribution (e.g., DAR=0-10) or a narrow distribution (e.g., DAR=3-4). For such mixtures, DAR often refers to the average DAR of the mixture. This is well known in the field of bioconjugation. However, if the conjugation occurs via a glycan (i.e., b=1 and L 6 is present), the antibody conjugate according to the invention has a DAR close to the theoretical DAR. For example, if the theoretical DAR is 4, DAR values ​​of greater than 3.6 or greater than 3.8 are readily obtained, indicating that most of the antibodies in the reaction mixture have reacted completely, resulting in a DAR of 4.

[0068] Linking group Z Z is a connecting group that covalently connects both parts of the conjugate according to the invention. The term "connecting group" in this specification refers to a structural element resulting from the reaction between Q and F that connects one part of a conjugate with another part of the same conjugate. As will be understood by those skilled in the art, the nature of the connecting group depends on the type of reaction that results in the connection between the parts of the compound. As an example, if 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' through the connecting group Z, and Z can be represented by the group -C(O)-N(H)-. The connecting group Z can take any form since it is derived from the reaction between Q and F.

[0069] Since more than one reactive moiety F may be present or introduced in the antibody, the antibody conjugate according to the invention may contain more than one payload D, such as 1 to 8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D per biomolecule. The number of payloads is typically an even number, taking into account the symmetrical nature of antibodies. In other words, if one side of the antibody is functionalized with F, the symmetrical counterpart will also be functionalized. Alternatively, if the naturally occurring thiol group of a cysteine ​​residue of a protein is used as F, the value of m can be any and can vary between individual conjugates.

[0070] In the compound according to structure (1), the connecting group Z is optionally connected to L via a linker L. 6 D is linked to AB via AB. Numerous reactions for attaching reactive group Q to reactive group F are known in the art. As a result, a wide variety of connecting groups Z may be present in the conjugates according to the invention. In one embodiment, the reactive group Q is preferably selected from the options above, as shown in FIG. 1, and the complementary reactive group F and the connecting group Z thus obtained are known to those skilled in the art. When a linker conjugate containing Q is conjugated to a biomolecule containing a complementary reactive group F, F and Q, as well as the connecting group Z, present in the bioconjugate, 3Some examples of suitable combinations are shown in FIG.

[0071] For example, when F contains or is a thiol group, the complementary group Q includes N-maleimidyl and alkenyl groups, and the corresponding connecting group Z is as shown in Figure 1. When F contains or is a thiol group, the complementary group Q also includes an arenamide group.

[0072] For example, when F includes or is an amino group, the complementary group Q includes a ketone group and an activated ester group, and the corresponding connecting group Z is as shown in FIG.

[0073] For example, when F contains or is a ketone group, the complementary groups Q include (O-alkyl)hydroxylamino groups and hydrazine groups, and the corresponding connecting groups Z are as shown in FIG.

[0074] For example, when F contains or is an alkynyl group, the complementary group Q contains an azide group and the corresponding connecting group Z is as shown in FIG.

[0075] For example, when F comprises or is an azide group, the complementary group Q comprises an alkynyl group and the corresponding connecting group Z is as shown in FIG.

[0076] For example, when F comprises or is a cyclopropenyl group, a transcyclooctene group, or a cyclooctyne group, the complementary group Q comprises a tetrazinyl group and the corresponding connecting group Z is as shown in Figure 1. In these particular cases, Z is merely an intermediate structure that expels N2, thereby generating a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0077] Further suitable combinations of F and Q, and the nature of the resulting connecting group Z3, are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), which is incorporated by reference, in particular in Chapter 3, pages 229 to 258. A list of complementary reactive groups suitable for bioconjugation processes is disclosed in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), Chapter 3, pages 230 to 232, Table 3.1, the contents of which are expressly incorporated herein by reference.

[0078] In a preferred embodiment, the linking group Z is obtained by a cycloaddition or nucleophilic reaction, preferably 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. Such a 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, who will be able to select the appropriate reaction partners F and Q and will understand the nature of the resulting linking group Z.

[0079] In a first preferred embodiment, Z is formed by cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reaction) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are inverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art and the skilled person knows how to carry them out.

[0080] Preferably, Z comprises 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. It is particularly preferred that a triazole moiety is present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e. is formed from Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. is formed from Q comprising a (hetero)cycloalkene moiety. In a preferred embodiment, Z has the structure (Z1). [ka]

[0081] Where: [ka] A bond shown as is a single bond or a double bond. the ring Z is obtained by cycloaddition, preferably the ring Z is selected from (Za) to (Zj) as defined below, ** The carbon atom labeled with represents the carbon atom of (Z1) to which ring Z is fused. [ka] corresponds to the two carbon atoms of the bond shown as -R 15 are independently 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 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -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 (+) is B (-) is a cationic sulfur atom offset by (-) is an anion, and each R 31 are individually, R 15or a connection to D connected via L, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; -v=an integer in the range of 8 to 16, Ring A is formed by cycloaddition and is preferably selected from (Za)-(Zj).

[0082] [ka] is a double bond, it is preferred that u+u'=4, 5, 6, 7, or 8. * The wavy bond labeled with is connected to S, ** The wavy bond labeled with is connected to L.

[0083] Z comprises a (hetero)cycloalkene moiety, i.e. [ka] It is particularly preferred that the bond shown as: is a double bond. In a preferred embodiment, Z is selected from structures (Z2) to (Z20) shown below: [ka]

[0084] Here, the connection to L is shown as a wavy coupling. (-)is an anion, preferably a pharma- ceutically 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) to (Zm) shown below, where: ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z2) to (Z20) to which the ring Z is fused. Preferred rings Z are selected from (Za) to (Zj), more preferably (Za), (Zd) and (Zh), and most preferably ring Z has the structure (Za). The connecting group Z is formed in the context of this embodiment by reaction with a (hetero)cycloalkyne, and therefore [ka] The bond shown above as is a double bond. [ka]

[0085] In a further preferred embodiment, Z is selected from structures (Z21) to (Z38) and (Z8a) shown below. [ka]

[0086] Here, the connection to L is shown as a wavy bond. In structure (Z38), B (-) is an anion, preferably a pharma- ceutically acceptable anion. Ring Z is selected from structures (Za) to (Zm) as defined above, preferably structures (Za) to (Zj).

[0087] In preferred embodiments, Z comprises a (hetero)cyclooctene or (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.

[0088] Thus, in a preferred embodiment, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, a heterocycloheptyne moiety according to structure (Z37) is unsubstituted.

[0089] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably a (hetero)cyclooctene moiety 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) shown below, where V is (CH2) l where 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 conforms to structure (Z42), further defined below.

[0090] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27), or (Z28), which are optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) shown below, where Y 1 is O or NR 11 where R 11 are independently hydrogen, linear or branched C1-C12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring in (Z40) is optionally O-sulfonylated at one or more positions, while the ring in (Z41) can be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), which is further defined below.

[0091] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and conforms to the structure (Z37). [ka]

[0092] In particularly preferred embodiments, Z comprises a cyclooctenyl group and conforms to the structure (Z42). [ka] Where: - * The bond labeled with is connected to S, ** The wavy bond labeled with is connected to L, -R 15 are independently 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 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 19 is hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group is optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted or R 19 is a second occurrence of Z (or Q) or D connected via a spacer moiety, -l is an integer in the range of 0 to 10.

[0093] In a preferred embodiment of a group according to structure (Z42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of a group according to structure (Z42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of a group according to structure (Z42), R 19 is H. In preferred embodiments of groups according to structure (Z42), I is 0 or 1, and more preferably, l is 1.

[0094] In particularly preferred embodiments, Z comprises a (hetero)cyclooctynyl group and conforms to the structure (Z43). [ka] Where: - * The bond labeled with is connected to S, ** The wavy bond labeled with is connected to L, -R 15 are independently 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 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y is N or CR 15 It is.

[0095] In a preferred embodiment of a group according to structure (Z43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of groups according to structure (Z43), Y is N or CH, more preferably Y=N.

[0096] In particularly preferred embodiments, Z comprises a heterocycloheptynyl group according to structure (Z37) or (Z38a), preferably according to structure (Z37), and ring Z is a triazole ring. [ka]

[0097] In an alternative preferred embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. [ka] The bond shown as is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkanyl group or a cycloalkanyl group, preferably a cycloalkanyl group, where the (hetero)cycloalkanyl group is optionally substituted. Preferably, the (hetero)cycloalkanyl group is a (hetero)cyclopropanyl group, a (hetero)cyclobutanyl group, a norbornane group, a norbornene group, a (hetero)cycloheptanyl group, a (hetero)cyclooctanyl group, a (hetero)cyclononyl group, or a (hetero)cyclodecanyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropanyl groups, (hetero)cycloheptanyl groups, or (hetero)cyclooctanyl groups, where the (hetero)cyclopropanyl group, the trans-(hetero)cycloheptanyl group, or the (hetero)cyclooctanyl group are optionally substituted. Preferably, Z comprises a cyclopropanyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornane or norbornene group according to structure (Z46), a (hetero)cycloheptanyl moiety according to structure (Z47), or a (hetero)cyclooctanyl moiety according to structure (Z48), where Y 3 is C(R 23 )2, NR 23 or O, 23 are individually hydrogen, C1-C6 alkyl, or are optionally connected to L via a spacer; [ka] The bond labeled with is a single bond or a double bond. In a further preferred embodiment, the cyclopropanyl group conforms to structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group conforms to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group conforms to structure (Z52), (Z53), (Z54), (Z55) or (Z56). [ka]

[0098] wherein the R group(s) on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is typically selected from structures (Zn)-(Zu), where: ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z44) to (Z56) to which the ring Z is condensed, * The carbon labeled with is directly connected to the peptide chain of the antibody. Preferred rings Z are selected from (Zo) to (Zr). The connecting group Z is formed in the context of this embodiment by reaction with a (hetero)cycloalkene, and therefore: [ka] The bond shown above as is a single bond. [ka]

[0099] In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably a nucleophilic substitution or a Michael addition, preferably a Michael addition. A preferred Michael reaction is a thiol-maleimide ligation, most preferably Q is a maleimide and F is a thiol group. Preferably, the thiol is present in the side chain of a cysteine ​​residue. In a preferred embodiment, the connecting group Z comprises a succinimidyl ring or a ring-opened succinamide derivative thereof. Preferred choices of the connecting group Z include moieties selected from (Z57) to (Z71) shown below. [ka]

[0100] Where: *The wavy bond(s) labeled with are optionally connected to the antibody Ab via a linker, and the unlabeled wavy bond is optionally connected to the payload via a linker. 29 is C 1~12 Alkyl, preferably C 1~4 is alkyl, most preferably ethyl; X 1 is O or S, preferably X 1 =O. (Z67)~(Z71) ** The nitrogen atom labeled with corresponds to the nitrogen atom of the side chain of a lysine residue in the antibody. The carbon atoms of the phenyl groups of (Z69) and (Z70) are optionally substituted and preferably optionally fluorinated.

[0101] In a preferred embodiment, the connecting group Z comprises a moiety selected from (Z1)-(Z71).

[0102] Linker L The linker L connects the payload D with the connecting group Z (in a conjugate according to structure (1)) or the payload D with the reactive group Q (in a compound according to structure (2)). Linkers are known in the art and may be cleavable or non-cleavable. The linker L preferably comprises a self-immolative group or a cleavable linker comprising a peptide spacer and a para-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0103] In a preferred embodiment, (L 4 ) q is connected to the payload D, (L 1 ) n is connected to Z or Q, structure -(L 1 ) n -(L 2 ) o -(L 3 ) p -(L 4 ) q - as a linker L, where L 1 , L 2 , L 3 , and L 4is a linker or linking unit, and each of n, o, p, and q is individually 0 or 1, and n+o+p+q is at least 1. In a preferred embodiment, at least the linker L 1 and L 2 is present (i.e., n=1, o=1, p=0 or 1, q=0 or 1), and more preferably, the linker L 1 , L 2 , and L 3 exists, and L 4 is present or absent (i.e., n=1, o=1, p=1, q=0 or 1). In one embodiment, the linker L 1 , L 2 , L 3 , and L 4 (i.e., n=1, o=1, p=1, q=1). In one embodiment, the linker L 1 , L 2 , and L 3 exists, and L 4 does not exist (i.e., n=1, o=1, p=1, q=0).

[0104] Linkers, especially linker L 1 may contain one or more branching points for attaching multiple payloads to a single connecting group. In a preferred embodiment, the linker of the conjugate according to the invention comprises a branching moiety. A "branching moiety" in the context of the present invention refers to a moiety embedded in a linker that connects three moieties. In other words, the branching moiety comprises at least three bonds to other moieties, typically one bond to Z or Q, one bond to payload D, and one bond to a second payload D. The branching moiety, when present, is preferably located at the linker L 1 More preferably, Sp 2 Part of, or NR 13 embedded as a nitrogen atom of. Any moiety that contains at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. In a preferred embodiment, the branched 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 branched moiety is a nitrogen atom.

[0105] Linker L 1 Linker L 1 is absent (n=0) or present (n=1). 1 exists and n=1. L 1 For example, linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, 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 The alkylene group may be selected from 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. Optionally, the alkylene group, the alkenylene group, the alkynylene group, the cycloalkylene group, the cycloalkenylene group, the cycloalkynylene group, the alkylarylene group, the arylalkylene group, the arylalkenylene group, and the arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, which are preferably O, S(O), y’ , and N.R. 21 wherein y′ is 0, 1, or 2, preferably y′=2; R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups.

[0106] In a preferred embodiment, the linker L 1contains a polar group. Such polar groups include (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 (where z' is the number of carbon atoms in the alkane, preferably z'=1-10), -(O) a -C(O)-NH-S(O)2-NR 13 - (see structure (23) as further defined below), -C(S(O)3 (-) )-, -C(C(O)2 (-) )-, -S(O)2-, -P(O)2 (-) -, -O(CH2CH2O) t -, -NR 30 (CH2CH2NR 30 ) t -, and may be selected from the following two structures: [ka]

[0107] The polar group may also preferably comprise an amino acid selected from Arg, Glu, Asp, Ser, and Thr, where a and R 13 is further defined below with respect to structure (23). t is an integer in the range of 0 to 15, preferably 1 to 10, more preferably 2 to 5, and most preferably t=2 or 4. Each R 30 are, respectively, H, C 1~12 Alkyl, C1~ 12 Aryl, C 1~12 Alkaryl or C 1~12 Aralkyl. Linker L 1 may contain two or more such polar groups, such as at least two polar groups. The polar groups may also be linked to linkers L, which branch the branching moieties as defined elsewhere. 1 Preferably, nitrogen or carbon atoms are used as branching moieties. In the branch, -O(CH2CH2O) tThe presence of polar groups is particularly preferred.

[0108] In a preferred embodiment, the linker L 1 is or contains a sulfamide group, preferably a sulfamide group according to structure (23). [ka]

[0109] The wavy lines represent the remainder of the compound, typically Q and L. 2 , L 3 , L 4 or D, preferably Q and L 2 Preferably, (O) a The C(O) moiety is connected to Q and is 13 The part is L 2 , L 3 , L 4 or D, preferably L 2 is connected to.

[0110] In structure (23), a=0 or 1, preferably a=1, R 13 is hydrogen, C 1~ C 24 Alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (Hetero)aryl groups, C 3~ C 24 Alkyl(hetero)aryl groups, and C 3~ C 24 (hetero)arylalkyl groups, 1~ C 24 Alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (Hetero)aryl groups, C 3~ C 24 Alkyl(hetero)aryl groups, and C 3~ C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR14 and R 14 are independently hydrogen and C 1~ C4 alkyl groups or R 13 is preferably linked via a spacer moiety to Sp 2 and in one embodiment, D is connected to N via -(B) e -(A) f -(B) g It is connected to N via -C(O)-.

[0111] In a preferred embodiment, R 13 is hydrogen or C1-C 20 More preferably, R 13 is hydrogen or C1-C 16 is an alkyl group, and even more preferably, R 13 is hydrogen or C1-C 10 alkyl groups, which are optionally substituted, including O, S, and NR 14 R is optionally interrupted by one or more heteroatoms, preferably O, selected from 14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 13 is hydrogen. In another preferred embodiment, R 13 is C1~C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 is an alkyl group, optionally interrupted by one or more O atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is further preferred that R is a (poly)ethylene glycol chain containing a terminal -OH group. 13is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl, and i-propyl, even more preferably from the group consisting of hydrogen, methyl, and ethyl. Even more preferably, R 13 is hydrogen or methyl, and most preferably R 13 is hydrogen.

[0112] In a preferred embodiment, L 1 follows the structure (24). [ka]

[0113] where a and R 13 is as defined above, and 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 are independently linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene groups, and C9-C 200 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. When the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, the groups are preferably interrupted by one or more O atoms and / or one or more SS groups.

[0114] More preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylene group, C3-C 100 Cycloalkylene group, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkyl arylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylalkenylene groups, and C9-C 100 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0115] Even more preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylene group, C3-C 50 Cycloalkylene group, C5-C 50 Cycloalkenylene group, C8-C 50 Cycloalkynylene group, C7-C 50 Alkyl arylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene groups, and C9-C 50 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0116] Even more preferably, the spacer moiety Sp 1 and Sp 2When present, each independently represents a linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylene group, C3-C 20 Cycloalkylene group, C5-C 20 Cycloalkenylene group, C8-C 20 Cycloalkynylene group, C7-C 20 Alkyl arylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene groups, and C9-C 20 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0117] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and include O, S, and NR 16 , optionally interrupted by one or more heteroatoms, preferably selected from the group of O, 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0118] Most preferably, the spacer moiety Sp1 and Sp 2 When present, each independently represents a linear or branched C1-C 20 alkylene groups, which are optionally substituted, O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is selected from the group consisting of O, S, and NR 16 , preferably O and / or SS, optionally interrupted by one or more heteroatoms, 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0119] Therefore, the preferred spacer moiety Sp 1 and Sp 2 is -(CH2) r -, -(CH2CH2) r -, -(CH2CH2O) r -, -(OCH2CH2) r -, -(CH2CH2O) r CH2CH2-, -CH2CH2(OCH2CH2) r -, -(CH2CH2CH2O) r -, -(OCH2CH2CH2) r -, -(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) rIn the formula, 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 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.

[0120] Alternatively, the preferred linker L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein - d = 0 or 1, preferably d = 1, - e = 0 to 10, preferably e = an integer in the range of 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; - f = 0 or 1, preferably f = 0, -d+e+f is at least 1, preferably in the range of 1 to 5, preferably d+f is at least 1, preferably d+f=1. - g = 0 or 1, preferably g = 1; - k = 0 or 1, preferably k = 1; -A is a sulfamide group according to structure (23), -B is a -CH2-CH2-O- or -O-CH2-CH2- moiety, or e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 a -CH2- moiety, where e1 is defined as e, -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) mC(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably W is -OC(O)NH-, -C(O)(CH2) m C(O)NH- or -C(O)NH-, where m is an integer in the range of 0 to 10, preferably m=0, 1, 2, 3, 4, 5 or 6, and most preferably m=2 or 3; Preferably, L 1 (W) k via Q, and (C(O)) g Preferably, L via C(O) 2 , L 3 , L 4 , or D, preferably L 2 is connected to.

[0121] In the context of this embodiment, the wavy line in structure (23) represents (W) k , (B) e , and (C(O)) g A preferably conforms to structure (23), where a=1 and R 13 =H or C1~C 20 Alkyl groups, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0122] Preferred linkers L 1 Structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein (a) k=0, d=1, g=1, f=0, B=-CH2-CH2-O-, and e=1, 2, 3, or 4, preferably e=2. (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. (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. (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. (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. (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. (g) k=0, d=0, g=1, f=0, B=-CH2-CH2-O-, and e=1, 2, 3, or 4, preferably e=2. (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.

[0123] In a preferred embodiment, the linker L 1 contains a branched nitrogen atom, which is a group consisting of Q or Z and (L 2 ) o and contains as a substituent a further moiety D, which is preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR 13 where R 13 is connected to the second occurrence of D via a spacer moiety. Alternatively, the branched nitrogen atom can be represented by the structure -(W) k -(A) d -(B) e -(A) f -(C(O))g -Following L 1 In one embodiment, L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -N * [-(A) d -(B) e -(A) f -(C(O)) g’ -]2, in which A, B, W, d, e, f, g, and k are as defined above and are individually selected for each occurrence; N * is -(A) d -(B) e -(A) f -(C(O)) g’ - are two examples of connected branched nitrogen atoms, where both (C(O)) g’ The part is -(L 2 ) o -(L 3 ) p -(L 4 ) q -D, wherein L 2 , L 3 , L 4 , o, p, q, and D are as defined above and are each individually selected. In a preferred embodiment, L 2 , L 3 , L 4 , o, p, q, and D are each (C(O)) g’ is the same for both parts connected to

[0124] Preferred linkers L containing a branched nitrogen atom 1 Structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -N * [-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, (i) k=d=g=e'=1, f=d'=g'=0, W=-C(O)-, B=B'=-CH2-CH2-O-, A is according to structure (23), a=0 and R 13 =H, and e=1, 2, 3, or 4, preferably e=2. (j) k=d=g=e'=g'=1, f=d'=0, W=-C(O)-, B=B'=-CH2-CH2-O-, A is according to structure (23), a=0 and R 13 =H, and e=1, 2, 3, or 4, preferably e=2.

[0125] Linker L 2 Linker L 2 is absent (o=0) or present (o=1). 2 is present and o=1. 2 is a peptide spacer. The peptide spacer is preferably (NH-CR 17 -CO) n wherein R 17represents an amino acid side chain as known in the art. As used herein, the amino acids may be natural or synthetic amino acids. Preferably, the amino acid(s) are all in their L-configuration. n is an integer in the range of 1 to 5, preferably in the range of 2 to 5. Thus, the peptide spacer preferably comprises 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3) or tetrapeptide (n=4), most preferably the peptide spacer is a dipeptide. Although any peptide spacer may be used, preferably the peptide spacer is 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-Lys , Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Gly-Gly-Phe-Gly and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn, most preferably Val-Cit or Val-Ala. As used herein, AcLys is acetyl lysine and iGlu is isoglutamate. In one embodiment, L 2 In one embodiment, L 2 =Val-Ala.

[0126] R 17represents 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 those 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. Alternatively, R 17 is preferably 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 embodiments of R are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). 17 is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0127] In a particularly preferred embodiment, the peptide spacer may be represented by the general structure (L3): [ka]

[0128] In the formula, R 17is as defined above, preferably R 17 is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates (L 1 ) n and (L 3 ) p and preferably according to the structure (L3). 2 is via NH (L 1 ) n through C(O) (L 3 ) p is connected to.

[0129] Linker L 3 Linker L 3 is absent (p=0) or present (p=1). 3 is present and p=1. 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is a paraaminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L4). [ka]

[0130] Here, the wavy lines represent Q or Z, L 1 Or L 2 , and L 4 or D. Typically, the PABC derivatives are connected to Q, Z, L, or D via NH. 1 , or L 2 , preferably L 2 to, and through O to L 4 Or connected to D.

[0131] A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Preferred 5-membered rings are oxazole, thiazole and furan. Preferred 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.

[0132] R 21 , H, R 26 , C(O)OH, and C(O)R 26 Selected from R 26 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, include O, S, and NR 28 and R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 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, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and 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 is H.

[0133] Linker L 4 Linker L 4 is absent (q=0) or present (q=1). 4 is present and q=1. 4 teeth, -Structure-NR 22 -(C z -alkylene)-C(O)-, where z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl; -Structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 an ethylene glycol spacer according to -C(O)-, where e6 is an integer ranging from 1 to 10, e7 is an integer ranging from 1 to 3, and R 22 is H or C1-C4 alkyl, and -Structure-NR 22 -(C z -alkylene)-NR 22 -(C(O)) h - a diamine spacer according to the formula (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.

[0134] Linker L 4 represents an aminoalkanoic acid spacer, i.e., -NR 22 -(C z -alkylene)-C(O)-, where z is an integer in the range of 1 to 20, preferably 1 to 10, and most preferably 1 to 6. As used herein, the aminoalkanoic acid spacer is typically linked to L via a nitrogen atom. 3 and to D via a carbonyl moiety. 4 is selected from 6-aminohexanoic acid (Ahx, z=5), β-alanine (z=2) and glycine (Gly, z=1), and even more preferably is 6-aminohexanoic acid or glycine.4= 6-aminohexanoic acid. In one embodiment, L 4 = glycine. 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0135] Alternatively, the linker L 4 Structure -NR 22 -(CH2-CH2-O) e6 -(CH2) e7 It may also be an ethylene glycol spacer according to -(C(O)-, where 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. In the present specification, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0136] Alternatively, the linker L 4 Structure -NR 22 -(C z -alkylene)-NR 22 -(C(O)) h -, where 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, and 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 is methyl. As used herein, h is preferably 1, in which case the linker L 4 is particularly suitable for conjugation via the phenolic hydroxyl groups present on the payload D.

[0137] Payload D D represents a targeting molecule D that is or will be attached to the antibody, also referred to in the art as a payload. D is exatecan.

[0138] Compounds according to general structure (2) may contain two or more moieties D. When two or more cytotoxins D are present, the cytotoxins D may be the same or different, typically they are the same. In a preferred embodiment, compounds according to general structure (2) contain one or two occurrences of D, most preferably two occurrences of D. Typically, the second occurrence of D is present in a linker L, which may contain a branched moiety, typically a branched nitrogen atom, connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branched moiety via the same linker. Similarly, antibody conjugates according to structure (1) may contain two or more moieties D per connecting group Z.

[0139] Preferred Antibody Conjugates A preferred antibody conjugate according to the first aspect is selected from the group consisting of compounds (I) to (III), more preferably (II) or (III), most preferably (II). A more preferred antibody conjugate is selected from (X) to (XIII). In one 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 (XIIIe). The structures of these antibody conjugates are defined below.

[0140] The antibody conjugate (I) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-(L 4 ) q -D}x ] y (I) Where: -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined above as -(A) d -(B) e -(A) f -(C(O)) g - is a linker represented by -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), -L 4 is -N-(C z -alkylene)-C(O)- or -NR 22 -(C z -alkylene)-NR 22 where z and R 22 is as defined above, -q=0 or 1.

[0141] In the context of the antibody conjugate (I), L 1 For d=1 (A according to structure (23), a=1 and R 13 =H), e=2, f=0 and g=1. In the context of the antibody conjugate (I), L 2 In the context of the antibody conjugate (I), L 3 For R 21 = H. In the context of antibody conjugate (I), when q=1, it is preferred that z=1 or 5.

[0142] In a particularly preferred embodiment, the antibody conjugate according to structure (I) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0143] The antibody conjugate (II) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-D} x ] y (II) Where: -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), where R 21 =H.

[0144] In the context of the antibody conjugate (II), L according to structure (23) 1 , e=2, for A, a=1 and R 13 In the context of the antibody conjugate (II), L 2 =Val-Cit is preferred.

[0145] In a particularly preferred embodiment, the antibody conjugate according to structure (II) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0146] The antibody conjugate (III) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-(L 4 )-D} x ] y (III) Where: -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), where R 21 =H, -L 4 -NR 22 -(C z -alkylene)-NR 22 -, wherein R 22 is as defined above, and z is an integer in the range of 1 to 6.

[0147] In the context of the antibody conjugate (III), L 1 For e=2, a=1 and R 13 In the context of the antibody conjugate (III), L 2In the context of the antibody conjugate (III), it is preferred that z=2.

[0148] In a particularly preferred embodiment, the antibody conjugate according to structure (III) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0149] The antibody conjugate (X) has a linker-payload portion according to the following structure: [ka] Where: - The wavy line indicates the connection to Z, -L 2 , o and D are as defined above.

[0150] L 2 may be absent, preferably L 2 is present and o=1. For preferred antibody conjugates (Xa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (Xb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (X) preferably has the structure (Xa).

[0151] In a particularly preferred embodiment, the antibody conjugate according to structure (X) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0152] The antibody conjugate (XI) has a linker-payload portion according to the following structure: [ka] Where: - The wavy line indicates the connection to Z, -L 2 , o and D are as defined above.

[0153] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (XI) preferably has the structure (XIb).

[0154] In a particularly preferred embodiment, the antibody conjugate according to structure (XI) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably calicheamicin as payload D.

[0155] The antibody conjugate (XII) has a linker-payload portion according to the following structure: [ka] Where: - The wavy line indicates the connection to Z, -L 2 , L 4 , o, q, and D are as defined above.

[0156] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2.

[0157] L 4 For the preferred antibody conjugate (XIIc), q=0 and L 4 For the preferred antibody conjugate (XIId), q=1 and L 4 Structure -NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0158] For the preferred antibody conjugate (XIIe), 2 follows the structure (L3), R 17 is CH3, q=0, and L 4 For the preferred antibody conjugate (XIIf),2 follows the structure (L3), R 17 is CH3, q=1, and L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, where z is an integer ranging from 1 to 10, preferably z=2, and R 22 is H or C1-C4 alkyl.

[0159] For the preferred antibody conjugate (XIIg), 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0, L 4 For the preferred antibody conjugate (XIIh), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0160] In the context of the antibody conjugate (XII), z is preferably an integer in the range of 1 to 10, more preferably z=2 to 6, and most preferably z=2. In the context of the antibody conjugate (XII), R 22 is more preferably H or CH3.

[0161] In the context of the antibody conjugate (XII), structures (XIIg) and (XIIh) are most preferred.

[0162] In a particularly preferred embodiment, the antibody conjugate according to structure (XII) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0163] The antibody conjugate (XIII) has a linker-payload portion according to the following structure: [ka] Where: - The wavy line indicates the connection to Z, -L 2 , L 4 , o, q, and D are as defined above.

[0164] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIIIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIIIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH3.

[0165] L 4 For the preferred antibody conjugate (XIIIc), q=0 and L 4 For the preferred antibody conjugate (XIIId), q=1 and L 4 Structure -NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R22 is H or C1-C4 alkyl.

[0166] For the preferred antibody conjugate (XIIIe), 2 follows the structure (L3), R 17 is CH3, q=0, and L 4 For the preferred antibody conjugate (XIIIf), L 2 follows the structure (L3), R 17 is CH3, q=1, and L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, where z is an integer ranging from 1 to 10, preferably z=2, and R 22 is H or C1-C4 alkyl.

[0167] For the preferred antibody conjugate (XIIIg), 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0, L 4 For the preferred antibody conjugate (XIIIh), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0168] In the context of the antibody conjugate (XIII), z is preferably an integer in the range of 1 to 10, more preferably z=2 to 6, and most preferably z=2. In the context of the antibody conjugate (XIII), R 22 is more preferably H or CH3.

[0169] In the context of the antibody conjugate (XIII), the structure (XIIIe) is most preferred.

[0170] In a particularly preferred embodiment, the antibody conjugate according to structure (XIII) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0171] In one particularly preferred embodiment, the antibody conjugate according to the invention conforms to structure (XIIIe) defined above, the antibody is hRS7 as defined above and the payload is exatecan.

[0172] These preferred antibody conjugates are further preferably conjugated via a glycan (i.e. b=1), more preferably a trimmed glycan (i.e. j=0). It is further preferred herein that S=GalNAc and w'=0. It is further preferred herein that the connecting group Z is formed by azide-alkyne cycloaddition, preferably the connecting group Z=(Z39), with ring Z=(Za) and V=CH2. It is further preferred herein that x=1. It is further preferred herein that y=2, more preferably x=1 and y=2.

[0173] In a most preferred embodiment, the antibody conjugate according to the invention conforms to structure (XIIIe) defined above, wherein the antibody is hRS7 as defined above, the payload is exatecan, where b=1, e=0, S=GalNAc, w'=0, connecting group Z=(Z39), ring Z=(Za) and V=CH2, x=1 and y=2.

[0174] Compounds conforming to general structure (2) The compound has the general structure (2): QLD (2) Where: -Q is a reactive moiety, - L is a linker connecting Z to D, D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof;

[0175] Compounds of general structure (2) may also be referred to as "linker-drug constructs" to include the linker L and payload D of the final conjugate. Compounds of general formula (2) may be prepared by those skilled in the art using standard organic synthesis techniques and as illustrated in the Examples. Linker L and payload D are defined above in the context of the conjugate according to structure (1).

[0176] Reactive moiety Q Compounds according to general structure (2) include a reactive moiety Q. In the context of the present invention, the term "reactive moiety" can refer to a chemical moiety that includes a reactive group, but also to the reactive group itself. For example, a cyclooctynyl group is a reactive group that includes a CC triple bond as a reactive group. Similarly, an N-maleimidyl group is a reactive group that includes a CC double bond as a reactive group. However, reactive groups, such as an azide reactive group, a thiol reactive group, or an alkynyl reactive group, can also be referred to as reactive moieties herein.

[0177] Q is S(F) xIn other words, Q is reactive to and complementary to F. As used herein, a reactive group is referred to as "complementary" to a reactive group when the reactive group selectively reacts with the reactive group, optionally in the presence of another functional group. 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 advantageously used in conjugation reactions in which a chemical reaction between Q and F occurs, thereby forming an antibody conjugate comprising a covalent connection between the payload D and the antibody.

[0178] The exact nature of Q and F depends on the type of conjugation reaction used. The skilled person will be able to select the appropriate combination of Q and F. Preferably, Q, and therefore also F, are reactive in a cycloaddition or nucleophilic reaction. Thus, Q preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety, more preferably Q is a click probe, a thiol-reactive moiety, or an amine-reactive moiety, and most preferably Q is a click probe. The click probe is reactive in a cycloaddition (click reaction), and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an alkene or alkyne moiety, more preferably the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne. Exemplary thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, alenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, vinylsulfones, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Exemplary amine-reactive moieties are selected from N-hydroxysuccinimidyl 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 an alkene moiety, an alkyne moiety, a thiol-reactive moiety or an amine-reactive moiety, more preferably an alkene moiety or an alkyne moiety, even more preferably an alkyne moiety. In the present specification, the alkene is preferably a (hetero)cycloalkene, and the alkyne is preferably a terminal alkyne or (hetero)cycloalkyne. Most preferably, Q is a cyclic (hetero)alkyne moiety. Each of these moieties is further defined below.

[0179] Thus, in a particularly preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, and the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group or a (hetero)cyclodecynyl group. In the present specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, and the (hetero)cyclooctynyl group is optionally substituted.

[0180] In particularly preferred embodiments, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and conforms to the structure (Q1). [ka] Where: [ka] is a double or triple bond, -R 15 are independently 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(hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -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 (+) is B (-) is a cationic sulfur atom offset by (-) is an anion, and each R 31 are individually, R 15 or a connection to D connected via L, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; -v is an integer in the range of 0 to 16.

[0181] Typically, v = (u + u') × 2 (the connection to L shown by the wavy coupling is Y 2 (when the connection to L represented by the wavy bond is via one of the carbon atoms) or [(u+u')×2]−1 (when the connection to L represented by the wavy bond is via one of the carbon atoms).

[0182] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and conforms to structure (Q1a). [ka] Where: -R 15 and Y 2 is as defined above, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=4, 5, 6, 7, or 8; -v is an integer in the range of 8 to 16.

[0183] In preferred embodiments, u+u'=4, 5, or 6, more preferably u+u'=5. In preferred embodiments, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0184] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2)-(Q20) and (Q20a) shown below. [ka]

[0185] Here, the connection to L, shown as a wavy bond, may be to any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) may have a connection to L or may contain a hydrogen atom or may be optionally functionalized. (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) , or I (-) and most preferably selected from B (-) teeth (-) In the conjugation reaction, B (-) In any case, B will exchange with anions present in the reaction mixture. (-)does not have to be a pharma- ceutically acceptable anion. When (Q19) is used for Q, the negatively charged counterion is preferably pharma- ceutically acceptable upon isolation of the conjugate according to the invention, so that the conjugate can be readily used as a drug.

[0186] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38) and (Q38a) shown below. [ka]

[0187] In structure (Q38), B (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) , or I (-) and most preferably selected from B (-) teeth (-) It is OTf.

[0188] In preferred embodiments, Q comprises a (hetero)cyclooctyne or (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 for Q is further defined below.

[0189] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also referred to as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is unsubstituted.

[0190] In an alternative preferred embodiment, Q comprises a cyclooctyne moiety according to structure (Q8), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), more preferably a cyclooctyne moiety according to (Q29), 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) shown below, where V is (CH2) l where 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 conforms to structure (Q42), further defined below.

[0191] In alternative preferred embodiments, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27), or (Q28), also referred to 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) shown below, where Y 1 is O or NR 11 where R 11 are independently hydrogen, linear or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring in (Q40) may be optionally O-sulfonylated at one or more positions, while the ring in (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 according to structure (Q43), further defined below.

[0192] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and conforms to the structure (Q37). [ka]

[0193] In particularly preferred embodiments, Q comprises a cyclooctynyl group and conforms to the structure (Q42). [ka] Where: -R 15 are independently 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 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 19 is hydrogen, halogen, C1-C24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group is optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted or R 19 is a second occurrence of Q or D connected via a spacer moiety, -l is an integer in the range of 0 to 10.

[0194] In a preferred embodiment of the reactive group according to structure (Q42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is 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), I is 0 or 1, and more preferably, l is 1.

[0195] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and conforms to the structure (Q43). [ka] Where: -R 15 are independently 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 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y is N or CR 15 It is.

[0196] In a preferred embodiment of the reactive group according to structure (Q43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of the reactive group according to structure (Q43), Y is N or CH, more preferably Y=N.

[0197] In particularly preferred embodiments, Q comprises a heterocycloheptynyl group and conforms to structure (Q37) or (Q38a), preferably structure (Q37). [ka]

[0198] In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, where the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene 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 (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, or trans-(hetero)cyclooctenyl groups, which 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), where Y 3 is C(R 23 )2, NR 23 or O, 23 are individually hydrogen, C1-C6 alkyl, or are optionally connected to L via a spacer; [ka] The bond labeled with is a single bond or a double bond. In a further preferred embodiment, the cyclopropenyl group conforms to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group conforms to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group conforms to structure (Q52), (Q53), (Q54), (Q55) or (Q56). [ka]

[0199] Here, the R group(s) on Si in (Q50) and (Q51) are typically alkyl or aryl, preferably C1-C6 alkyl.

[0200] In an alternative preferred embodiment, Q is a thiol-reactive probe. In this embodiment, Q is a reactive group that is compatible with cysteine ​​conjugation. Such probes are known in the art and can be selected from the group consisting of maleimide moiety, haloacetamide moiety, alenamide moiety, phosphonamidite moiety, cyanoethynyl moiety, vinylsulfone, vinylpyridine moiety, or methylsulfonylphenyloxadiazole moiety. Most preferably, Q comprises or is a maleimide moiety. The reagent can be mono-alkylated or can be a crosslinker for reaction with two cysteine ​​side chains.

[0201] In a further preferred embodiment, probe Q is selected from the group consisting of (Q57) to (Q71) shown below. [ka] Where: -X 6 is H, halogen, PhS, MeS, preferably halogen such as Cl, Br, I, -X 7is a halogen, such as PhS, MeS, preferably Cl, Br, I, etc. -R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 is alkyl, -R 25 , H, C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkaryl or C 1~12 aralkyl, preferably H or paramethylphenyl; wherein the aromatic rings in (Q61) and (Q63) may optionally be heteroaromatic rings, such as phenyl or pyridine rings.

[0202] In a preferred embodiment of the thiol-reactive probe (Q57), probe Q is selected from the group consisting of (Q72) to (Q74) shown below. [ka] Where: -R 27 is C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkaryl or C 1~12 It is aralkyl, -t is an integer in the range of 0 to 15, preferably 1 to 10.

[0203] In an alternative 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-hydroxysuccinimidyl groups, isocyanate groups, isothiocyanate groups, and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimidyl ester or p-nitrophenyl carbonate moiety.

[0204] In a further preferred embodiment, probe Q is selected from the group consisting of (Q75) to (Q79) shown below. [ka] where X 2 is a halogen, preferably F.

[0205] In a preferred embodiment, Q is selected from the group consisting of (Q1) to (Q79).

[0206] Antibodies conforming to general structure (3) The antibody has the general structure (3): AB-[(L 6 ) b -{F} x ] y (3) Where: -AB is an antibody capable of targeting Trop-2-expressing tumors, -b is 0 or 1, -L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which 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, 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-, -F is a reactive moiety, -x is 1 or 2, -y is 1, 2, 3, or 4.

[0207] The antibody of general structure (3) may also be referred to as a "(modified) antibody" since it is an antibody comprising a reactive group F, which is naturally occurring or the antibody has been modified to incorporate a reactive group F. The (modified) antibody according to general formula (2) may be prepared by one of skill in the art using standard organic and / or enzymatic synthesis techniques and as illustrated in the examples. 6 , b, x and y are defined above in the context of the conjugate according to structure (1).

[0208] Reactive moiety F F is reactive to Q in a conjugation reaction defined below, preferably the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. As the skilled person will appreciate, the options for F are the same as the options for Q, except that F and Q are reactive to each other. Thus, F preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety, more preferably F is a click probe, a thiol or an amine, and most preferably F is a click probe. The click probe is reactive in a cycloaddition (click reaction), and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an orthoquinone, a dioxothiophene, a sydnone, an alkene moiety, and an alkyne moiety. Preferably, the click probe comprises or is an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an orthoquinone, a dioxothiophene, or a sydnone, most preferably an azide. Exemplary thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, alenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, orthoquinone moieties, vinylsulfones, vinylpyridines, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Exemplary amine-reactive moieties are selected from N-hydroxysuccinimidyl esters, isocyanates, isothiocyanates, and benzyl halides. In a preferred embodiment, F is a click probe or thiol, more preferably F is an azide or thiol, and most preferably F is an azide.

[0209] Two or more reactive groups F may be present in the antibody. The reactive groups F in the antibody may be naturally occurring or may be placed in the antibody by certain techniques, such as (bio)chemical or genetic techniques. The reactive groups placed in the antibody are prepared by chemical synthesis, such as azides or terminal alkynes. Methods for preparing modified antibodies are known in the art, for example from WO2014 / 065661, WO2016 / 170186 and WO2016 / 053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between modified antibodies and linker-drug constructs is known to those skilled in the art.

[0210] Preferably, F is a click probe reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and sydnones. Preferred structures of the reactive group are structures (F1) to (F10) shown below. [ka]

[0211] Here, the wavy bonds represent connections to the payload. For (F3), (F4), (F8), and (F9), the payload can be connected to any one of the wavy bonds. The other wavy bonds can then be connected to hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl groups, 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 a sulfonyl group, each of which (except hydrogen) may be optionally substituted, and optionally O, S, and NR 32 and R32 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. One of ordinary skill in the art will understand which R groups may apply to each of the groups F. For example, the R group linked to the nitrogen atom of (F3) may be selected from alkyl and aryl, and the R group linked to the carbon atom of (F3) may 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.

[0212] In a second preferred embodiment, F is a thiol or a precursor thereof. The thiol or a precursor thereof F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive to a thiol-reactive probe Q in a thiol ligation. The thiol is preferably the thiol of the side chain of a cysteine ​​amino acid naturally occurring in the antibody AB, in which case the linker L 6 is absent (b=0), but optionally includes a linker L 6 Thiol precursors in the context of bioconjugation are known in the art and include disulfides which may be naturally occurring disulfide bridges present in antibodies, or synthetically introduced disulfides which are reduced as known in the art. Preferably, F is a thiol group of a cysteine ​​side chain.

[0213] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or a precursor thereof F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive to the amine-reactive probe Q in a nucleophilic displacement. The amine is typically a primary amine, preferably the amine of the side chain of a lysine amino acid naturally occurring in the antibody AB, in which case the linker L 6 is absent (b=0), but optionally includes a linker L 6Preferably, F is a primary amine group of a lysine side chain.

[0214] Process for synthesizing antibody conjugates according to general structure (1) In a further aspect, the present invention relates to a process for the preparation of an antibody conjugate according to the invention, which process comprises a step of reacting Q of a compound according to the invention with a reactive group F of an antibody. Compounds according to general structure (2) and preferred embodiments thereof are described in more detail above. The process is carried out under conditions such that Q reacts with F to covalently link the antibody AB (3) to the payload D. In the method according to the invention, Q reacts with F to form a covalent bond between the antibody and the compound according to the invention. Complementary reactive groups Q and reactive groups F are known to the skilled person and are described in more detail below.

[0215] Any conjugation technique known in the art can be used to prepare the multifunctional antibody construct of the present invention.Suitable conjugation techniques include thiol ligation, lysine ligation, 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 reaction and cycloaddition, preferably, cycloaddition is [4+2] cycloaddition or [3+2] cycloaddition, and nucleophilic reaction is Michael addition or nucleophilic substitution. Suitable conjugation techniques are disclosed, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), WO2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, PCT / EP2021 / 050594, PCT / EP2021 / 050598 and NL2026947.

[0216] Thus, in a preferred embodiment of the conjugation process according to the invention, the conjugation is achieved via a nucleophilic reaction, such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is the acylation of a primary amino group with an activated ester. A preferred Michael reaction is the maleimide-thiol reaction, which is widely used in bioconjugation.

[0217] Thus, in a preferred embodiment of the conjugation process according to the present invention, the conjugation is achieved via cycloaddition. A preferred cycloaddition is a (4+2)-cycloaddition (e.g., Diels-Alder reaction) or a (3+2)-cycloaddition (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art and the skilled person knows how to carry them out.

[0218] The process according to this embodiment preferably relates to 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 the skilled person will know how to carry them out.

[0219] Thus, the process for preparing an antibody conjugate according to the invention according to this embodiment comprises reacting a modified antibody of structure (3) with a compound according to structure (2) to obtain an antibody conjugate of structure (1).

[0220] In a preferred embodiment, the process for preparing an antibody-conjugate according to the invention comprises the steps of: (i) an antibody containing y core N-acetylglucosamine (GlcNAc) moieties (where y=1, 2, 3, or 4) is reacted with an antibody of formula S(F) in the presence of a catalyst x -P(in the formula, S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside mono- or diphosphate, and the catalyst is S(F) x (capable of transferring a moiety to the core-GlcNAc moiety), AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) During the ceremony, -AB is an antibody capable of targeting Trop-2-expressing tumors, -Fuc is fucose, - obtaining a modified antibody according to (ii) coupling the modified antibody to the structure (2): QLD (2) During the ceremony, -Q is a reactive moiety, - L is a linker connecting Z to D, -D is reacted with a compound according to the formula (I) selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; and obtaining an antibody conjugate according to structure (1).

[0221] Step (i) In step (i), an antibody containing one, two, three, or four core N-acetylglucosamine moieties is reacted with an antibody of formula S(F) in the presence of a catalyst.x -P, wherein S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside monophosphate or diphosphate, and the catalyst is S(F) x The moiety can be transferred to a core-GlcNAc moiety. As used herein, the antibody is typically an antibody that has been trimmed to a core-GlcNAc residue, as further described below. Step (i) provides a modified antibody according to formula (26).

[0222] The starting material, i.e., an antibody comprising a core-GlcNAc substitution, is known in the art and can be prepared by known methods by the skilled artisan. In one embodiment, the process according to the invention further comprises deglycosylating the antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substitution, the core N-acetylglucosamine and the core N-acetylglucosamine substitution being optionally fucosylated. Depending on the nature of the glycan, 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 combinations thereof, the selection of which depends on the nature of the glycan. EndoSH is described in PCT / EP2017 / 052792, which is incorporated herein by reference, see Examples 1-3 and SEQ ID NO:1.

[0223] The structural features S and x are defined above for the antibody-conjugates according to the invention and apply equally to this embodiment. x The formula S(F) to be connected to xCompounds of -P are known in the art, e.g., 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 WO2009 / 102820, all of which are incorporated herein by reference, but some compounds S(F) x In a preferred embodiment, S(F)-P and their synthesis are disclosed. 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), and most preferably P=UDP. Preferably, S(F)xP 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-(buta-3-amino acid)-2-deoxy-galactose-UDP. Most preferably, S(F)xP 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-(buta-3-amino acid)-2-deoxy-galactose-UDP. x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0224] SCIENCE FICTION) x Suitable catalysts capable of transferring a moiety to a core-GlcNAc moiety are known in the art. Suitable catalysts are those that are capable of transferring a moiety to a core-GlcNAc moiety, such as a specific sugar derivative nucleotide S(F) in that particular process. x-P is a catalyst for which the substrate. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcT) and β(1,4)-galactosyltransferases (GalT), and most preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases having a mutated catalytic domain. Suitable catalysts and mutations thereof are disclosed in WO2014 / 065661, WO2016 / 022027, and WO2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. The mutant enzymes described in WO2016 / 022027 and WO2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts are capable of recognizing internal sugars and sugar derivatives as acceptors. Thus, the sugar derivative S(F) x is linked to the core GlcNAc substituent in step (i), regardless of whether that GlcNAc is fucosylated or not.

[0225] 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.

[0226] Step (ii) In step (ii), the modified antibody is reacted with a compound according to general structure (2) comprising a reactive group F and a reactive group Q capable of reacting with a payload D to obtain an antibody conjugate according to structure (1) comprising a linking group Z resulting from the reaction between Q and F. Such reaction occurs under conditions such that 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.

[0227] 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 between a molecule containing an azide and a molecule containing a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the reactions known in the art as "click chemistry". In the case of a linker conjugate containing 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 contains a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required and the reaction can occur spontaneously by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry."

[0228] application The present invention relates to a method of treating a subject in need of treatment, comprising administering an antibody conjugate according to the invention as defined above. The subject in need of treatment is typically a cancer patient. The use of antibody conjugates, such as antibody drug conjugates, is well known in the field of cancer treatment, and the antibody conjugate according to the invention is particularly suitable in this respect. The described method is typically suitable for the treatment of cancer. In the method according to this aspect, the antibody conjugate is typically administered in a therapeutically effective amount. This aspect of the invention can also be expressed as an antibody conjugate according to the invention for use in the treatment of a subject in need of treatment, preferably for the treatment of cancer. In other words, this aspect relates to the use of an antibody conjugate according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of a subject in need of treatment, preferably for the treatment of cancer. In this context, it is envisaged that the treatment of cancer encompasses the treatment, imaging, diagnosis, prevention, inhibition and reduction of tumor growth.

[0229] This aspect of the invention may also be expressed as a method for targeting Trop-2 expressing cells, particularly Trop-2 expressing tumor cells, comprising contacting an antibody conjugate according to the invention with cells that may be CEA expressing. Thus, the method according to this aspect is suitable for determining whether a cell is Trop-2 expressing. 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 Trop-2 expressing cells preferably comprises one or more of treating, imaging, diagnosing, preventing, inhibiting and reducing the growth of Trop-2 expressing cells, particularly Trop-2 expressing tumor cells. The method according to this embodiment may be medical or non-medical. Non-medical methods according to this aspect may involve in vitro or ex vivo targeting of Trop-2 expressing cells, where the cells that may be Trop-2 expressing are present, for example, in a sample taken from a patient. Such non-medical methods are typically used to diagnose cancer, particularly Trop-2 positive cancers.

[0230] In the context of the present invention, the subject may suffer from a disorder selected from oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer, and lung cancer. Thus, the treatment of a subject in need thereof preferably refers to the treatment of oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer, and lung cancer.

[0231] The inventors have surprisingly found that the antibody conjugates according to the invention are superior in terms of safety and / or efficacy to conventional antibody conjugates targeting Trop-2, such that the therapeutic index of the antibody conjugates according to the invention is increased compared to conventional antibody conjugates targeting Trop-2.

[0232] Conjugation Style In the context of the present invention, "conjugation mode" refers to the process used to conjugate the payload D to the antibody AB, as well as the structural features of the resulting antibody conjugate, in particular the linker connecting the payload to the antibody, that are a direct result of the conjugation process. Thus, in one embodiment, the conjugation mode refers to the process for conjugating the payload to the antibody. In an alternative embodiment, the conjugation mode refers to the structural features of the linker and / or the attachment point of the linker to the antibody that are a direct result of the process for conjugating the payload to the antibody.

[0233] In a further aspect, the present invention relates to the use of a conjugation modality for increasing the therapeutic index of an antibody conjugate in the treatment of Trop-2 expressing tumors, the conjugation modality being used to connect an antibody AB with a payload D via a linker L. (i) an antibody containing y core N-acetylglucosamine (GlcNAc) moieties (where y=1, 2, 3, or 4) is reacted with an antibody of formula S(F) in the presence of a catalyst x -P(in the formula, S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside mono- or diphosphate, and the catalyst is S(F) x (capable of transferring a moiety to the core-GlcNAc moiety), AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) During the ceremony, -AB is an antibody capable of targeting Trop-2-expressing tumors, -Fuc is fucose, - obtaining a modified antibody according to (ii) coupling the modified antibody to the structure (2): QLD (2) During the ceremony, -Q is a reactive moiety, - L is a linker connecting Z to D, -D is reacted with a compound according to the formula (I) selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; and obtaining an antibody conjugate according to structure (1).

[0234] Preferably, increasing the therapeutic index of the antibody conjugate comprises: (a) increasing the therapeutic efficacy of the antibody conjugate, and / or (b) is selected because it increases the tolerability of the antibody conjugate.

[0235] The increased therapeutic efficacy of the antibody conjugate according to the invention may take the form of a reduced tumor size and / or a prolonged period of regression when compared to conventional Trop-2-targeted ADCs. The increased tolerability of the antibody conjugate according to the invention may take the form of a reduced sign of toxicity when compared to administration of a Trop-2-targeted ADC made by conventional techniques. The reduced 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 reduced weight loss, reduced mobility reduction, reduced food intake reduction, and / or one or more toxicity parameters such as improved blood chemistry, hematology, and / or histopathology. EXAMPLES

[0236] General procedure for transient expression and purification of monoclonal antibodies: Various IgGs (hRS7 and hTINA) were transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at a 300 mL scale. The supernatant was purified using a HiTrap MabSelect sure column. The supernatant was loaded onto the column and then washed with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl (TBS). The retained proteins were eluted with 0.1 M AcOH (pH 2.7). The eluted product was immediately neutralized with 2.5 M Tris-HCl pH 8.8 and dialyzed against 20 mM histidine, 150 mM NaCl, pH 7.5. The IgGs were then concentrated (>20 mg / mL) using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). The sequences of the IgGs are shown below.

[0237] hRS7(I) light chain (SEQ ID NO:3): DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0238] hRS7(I) heavy chain (SEQ ID NO:4): QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0239] hTINA(II) light chain (SEQ ID NO:5): DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0240] hTINA(II) heavy chain (SEQ ID NO:6): QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0241] General procedure for analytical RP-UPLC (DTT-treated samples): Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL 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 min. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD. Samples (5 μL) were injected at 0.4 mL / min onto a Bioresolve RP mAb2.1 × 150 mm 2.7 μm (Waters) at a column temperature of 70 °C. A linear gradient was applied from 30 to 54% acetonitrile in 0.1% TFA and water in 9 min. The absorbance of the eluted peak was measured at 215 nm followed by automatic integration (MassLynx, Waters) to determine reaction conversion.

[0242] General procedure for mass spectrometry analysis of (modified) monoclonal antibodies: Prior to mass spectrometry, IgG was treated with IdeS, which allows the analysis of Fc / 2 fragments. For the analysis of both light and heavy chains, a solution of 20 μg of (modified) IgG was incubated for 5 min at 37° C. with 100 mM DTT in a total volume of 4 μL. Azide functions, if present, are reduced to amines under these conditions. For the analysis of Fc / 2 fragments, a solution of 20 μg of (modified) IgG was incubated for 1 h at 37° C. with IdeS / Fabricator™ (1.25 U / μL) in phosphate buffered saline (PBS) pH 6.6 in a total volume of 10 μL. After dilution of the samples to 80 μL, electrospray ionization time of flight (ESI-TOF) was analyzed on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0243] General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg / mL) was incubated with 1% w / w EndoSH (see Examples 1-3 and SEQ ID NO: 1 as described in PCT / EP2017 / 052792, which is incorporated herein by reference), 3% w / w His-TnGalNAcT (see Examples 3 and 4 and SEQ ID NO: 49 as described in PCT / EP2016 / 059194, which is incorporated herein by reference), 6 mM MnCl2 and 0.01% AP (Roche) and UDP6-N3-GalNAc (compound 2d in Figure 3, 25 equivalents compared to IgG) in TBS for 16 hours at 30°C. The functionalized IgG was then purified using a HiTrap MabSelect sure 5mL column. After loading the reaction mixture, the column was washed with TBS+0.2% Triton and TBS. IgG was eluted with 0.1 M AcOH (pH 2.7) and neutralized with 2.5 M Tris-HCl pH 8.8. After three dialysis into 20 mM histidine, 150 mM NaCl pH 7.5, IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0244] Preparation of azide-functionalized antibodies: Example 1: Preparation of hRS7-(6-N3-GalNAc)2(I-N3) Following the general procedure for enzymatic remodeling, hRS7 was converted to hRS7-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 24360 Da, ∼80% of total Fc / 2) corresponding to the expected product.

[0245] Examples 2-4: Synthesis of linker conjugates 3 and 9 [ka] Example 2: Preparation of Compound 11 Compound 10 (163 mg, 240 μmol) was added to a mixture of exatecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMF (0.9 mL). After 20 h, the reaction mixture was diluted with 9 mL of DCM and purified by gradient column chromatography (0→40% MeOH / DCM) to give 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 FN6O 10 + (M+H) + Calculated 977.39, found 977.72. In addition to 11, exatecan free base (82.4 mg, 189 μmol, 20%) was recovered. 24 H 23 FN3O4 + (M+H) + The calculated value was 436.46 and the actual value was 436.54.

[0246] Example 3: Preparation of Compound 3 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 pale yellow solid (94 mg, 44 μmol, 28%). LCMS (ESI+)C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + The calculated value was 1066.88, and the actual value was 1067.12. [ka]

[0247] Example 4: Preparation of Compound 9 A solution of BCN-HS-PEG2-b-(Glu(OFm)-OH)2 (8, 12.1 mg, 10 μmol, 1.0 equiv.) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-exatecan (5b, Fmoc-deprotected 5, 19 mg, 25 μmol, 2.5 equiv.) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 equiv.) and HATU (8.9 mg, 23 μmol, 2.3 equiv.). After stirring at room temperature for 2 h, the reaction mixture was further diluted with DCM (800 μL) and purified by flash column chromatography on silica gel (0%→20% MeOH in DCM) to give the product as a clear oil (the presence of DMF makes it difficult to determine the yield). LCMS (ESI+)C 140 H 150 F2N 17 O 33 S + (M / 2+H + ) calculated value 1334.01, actual value 1334.79.

[0248] This compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 equiv.) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by flash column chromatography over silica gel (0%→45% MeOH in DCM) to give compound 9 as a yellow solid in 44% yield (10.2 mg, 4.4 μL). LCMS (ESI+)C 112 H 130 F2N 17 O 33 S + (M / 2+H + ) calculated value 1156.2, actual value 1156.74.

[0249] Examples 5-7: Conjugation of linker payload to (modified) monoclonal antibodies Example 5: Conjugation of hRS7(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PABC-Ex)23 to obtain conjugate hRS7-3 To a solution of hRS7(6-N3-GalNAc)2 (764.5 μL, 15.0 mg, 19.62 mg / ml in TBS pH 7.5) was added sodium deoxycholate (150 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (50 μL, 10 mM solution in DMF) and propylene glycol (400 μL). The reaction was incubated overnight at room temperature. To remove excess free payload, 15 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated for 2 hours. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (pore 0.20 μm, Corning). The solution was then buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and equilibrated with PBS. Free payload levels were still too high, so an additional 10 mg of activated charcoal was added and incubated overnight. The charcoal was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pores, Corning). The solution was then buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26497 Da, approximately 90% of total Fc / 2) corresponding to the conjugate hRS7-3. RP-UPLC analysis of the sample after IdeS treatment showed an average DAR of 3.90.

[0250] Example 6: Reduction of hTINA and conjugation with deruxtecan To a solution of hTINA (1780 μL, 10 mg, 5.63 mg / mL in PBS pH 7.4), PBS (27 μL), EDTA (40 μL, 500 mM), and TCEP (153 μL, 1 mM, 2.3 eq.) were added. The mixture was incubated at room temperature for 90 min. Conjugation was then performed by adding deruxtecan (200 μL, 1.8 mM, Achemblock) and incubating at room temperature for 90 min. The solution was then buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with PBS pH 7.4. The solution was then buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, equilibrated with 20 mM histidine, 6% sucrose pH 6.0, and 0.04% Tween-20, and then filter-sterilized. RP-UPLC analysis of the reduced conjugate showed an average DAR of 3.97.

[0251] Example 7: Conjugation of hRS7(6-N3-GalNAc)2 with BCN-HS-PEG2-(eva-PAB-Ex)29 to obtain conjugate hRS7-9 To a solution of hRS7(6-N3-GalNAc)2 (22.8 mL, 448 mg, 19.64 mg / ml in TBS pH 7.5) was added sodium deoxycholate (4.48 mL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)29 (895 μL, 10 mM solution in DMF) and propylene glycol (8.1 mL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). To remove excess free payload, 100 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated for 2 h. The charcoal was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore size, Corning). The solution was then dialyzed into 20 mM histidine, 6% sucrose buffer (pH 6.0). The material was concentrated and 0.04% Tween-20 was added before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26676 Da, approx. 90% of total Fc / 2) corresponding to the conjugate hRS7-9. RP-UPLC analysis of the sample treated with the fabricator (IdeS) showed an average DAR of 3.86.

[0252] Examples 8-9: In vivo testing Example 8: In vivo efficacy studies (performed at Charles River, Morrisville, NC, USA) 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.

[0253] CB.17 SCID mice (female), 8–12 weeks of age at the start of the study, were treated with 1 × 10 mice in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 7 Tumor cells were injected subcutaneously in the flank area. 3 Once the mice reached an average size of 100 mm, they were randomized into 3 groups of 8 mice and treatment was initiated.

[0254] Administration of the test article was via intravenous injection, with a dose volume of 10 mL / kg. Treatment began on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0255] After tumor cell inoculation, animals were checked daily for morbidity and mortality. At routine monitoring time points, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes were measured every 3-4 days in two dimensions using calipers, and volumetric data were expressed in mm3 using the formula V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. Experimental endpoints were determined using a 30-well plate with a diameter of 800 mm. 3 or greater than 20% tumor volume, or weight loss over 45 days, whichever occurs first. [Table 1]

[0256] Data for efficacy studies using the above test items are shown in Figures 8A and 8B. Figure 8A shows in vivo efficacy data for the NCI-N87 tumor model over time. hRS7-3 results in tumor regression. Tumor regression is similar to Trodelvy, but it is administered more frequently and at a higher dose level. Administration of HTINA-deruxtecan results in tumor growth delay. Figure 8B shows mouse body weight over time.

[0257] Example 9: In vivo efficacy studies Colo-205, a colon cancer xenograft model cell line, was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in a standard 5% CO2 humidified cell culture incubator at 37°C. Exponentially growing cells were harvested and counted for tumor inoculation.

[0258] BALB / c nude mice (female) aged 7–9 weeks were treated with 5 × 10 cells in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 6 Tumor cells were injected subcutaneously into the right anterior flank region. 3 Once the average size of the mice reached, they were randomized into 5 groups of 8 mice and treatment was initiated. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0259] Electronic caliper measurements were taken twice weekly. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice weekly. The experimental endpoints were 3,000 mm 3 tumor volume of 0.01 mg / kg or greater, or weight loss over 77 days, whichever occurs first.

[0260] Randomized: Mean tumor size approximately 143 mm 3 Randomization was performed once the tumor marker concentration reached 0. A total of 40 mice were enrolled in the NCI-H446 model study and randomly assigned to 5 groups with 8 mice per group. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0. Due to the cachectic nature of the tumor model, all animals received supplemental gel from the day of randomization.

[0261] Administration of test articles: Test articles were administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment began on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0262] Observations and Data Collection: After tumor cell inoculation, animals are checked daily for morbidity and mortality. At the time of routine monitoring, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using calipers, and volumetric data are expressed in mm using the formula V = (L x W x W) / 2. 3 where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. [Table 2]

[0263] Tumor volumes and mouse body weights over time in the efficacy study and corresponding Kaplan-Meier plots using the test parameters listed above are shown in Figures 9A, 9B and 9C.

[0264] Examples 10-12: In vitro testing Example 10. Human plasma stability study The stability of the ADC in human plasma was tested. Prior to the assay, plasma was depleted of all IgG using CaptivA® Protein A agarose (1 mL agarose / mL serum). ADC was added to the depleted human serum to a final concentration of 0.1 mg / mL followed by incubation at 37°C. At each time point, 0.5 mL was flash frozen and stored at -80°C until further analysis. To isolate the ADC after incubation, 20 μl of CaptivA® Protein A agarose resin was added to the sample and incubated for 1 h at room temperature. The resin was washed three times with PBS followed by the addition of 0.1 M glycine-HCl (pH 2.7) (0.4 mL) to elute the ADC. After elution, the sample was immediately neutralized with 1.0 M Tris pH 8.0 (0.1 mL). Samples were spin filtered three times against PBS using Amicon Ultra spin filters 0.5 mL MWCO 10 kDa (Merck Millipore) to reduce the volume to 40 μL, resulting in a final ADC concentration of approximately 1 mg / mL. Samples were analyzed by RP-UPLC (DTT reduction) on specific days to determine the DAR, the results of which are shown in the table below. At t=7 days, the difference from t=0 day is shown as a percentage. [Table 3]

[0265] Example 11. Thermal stability under physiological and enhanced stress conditions The stability of the ADCs was tested at elevated temperatures in either physiological conditions (PBS, pH 7.4, 37°C) or enhanced stress conditions (citrate buffered saline, CBS, pH 5.0, 40°C). The ADCs were buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with either PBS or CBS on an AKTA Pure (Cytiva). The solutions were concentrated to a concentration of >1 mg / mL using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius). The concentrations of the ADCs were measured and they were diluted to 1 mg / mL, and the first measurement, t=0, was taken for SE-HPLC analysis as described above. Samples were placed at either 37°C or 40°C and samples were taken at several time points (days) to determine aggregation levels. See Tables 4-6 below. [Table 4] [Table 5] [Table 6]

[0266] Example 12: HIC-HPLC measurements for determining relative retention times Samples were diluted to 1 mg / mL in PBS. HIC analysis is performed on an Agilent 1200 series using a TSKgel® Butyl-NPR HPLC column (3.5 cm x 4.6 mm, 2.5 μm). 10 μL of sample is injected at a flow rate of 0.5 mL / min in 20 min with a gradient starting from 100% Buffer A (2 M ammonium sulfate in 50 mM potassium phosphate pH 6.0) to 100% Buffer B (50 mM potassium phosphate pH 6.0 + 20% isopropanol). Native mAb (hRS7 or hTINA) is measured and its retention time is set to 1. Other measured samples are relative retention times compared to native mAb. The results are shown in Figure 10 and Table 7 below. [Table 7]

Claims

1. An antibody conjugate conforming to the general structure (1), AB-[(L ６ ) ｂ -{Z-L-D} ｘ ] ｙ （１） During the ceremony, -AB is an antibody that can target Trop-2 expressing tumors. -L is a linker that connects Z to D, -Z is a connecting group, -L ６ is -GlcNAc(Fuc) ｗ -(G) ｊ -S-(L ７ ) ｗ’ - and in the formula, 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, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L ７ is -N(H)C(O)CH ２ -, -N(H)C(O)CF ２ -, or -CH ２ - and -D is exatecan, -b is either 0 or 1, -x is either 1 or 2, -y is an antibody conjugate where y is 1, 2, 3, or 4.

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

3. The antibody has at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 and 5. Ｌ A domain and a V having at least 70% sequence identity with a sequence selected from the group consisting of sequence numbers 2 and 6. Ｈ The antibody conjugate according to claim 1, comprising a domain.

4. The antibody conjugate according to claim 1, wherein the antibody is hRS7 and / or comprises a light chain sequence according to SEQ ID NO: 3 and a heavy chain sequence according to SEQ ID NO: 4, and the sequence identity is at least 90%.

5. The antibody conjugate according to claim 1, 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, and the sequence identity is at least 90%.

6. Linker L is structure - (8) １ ) ｎ - (8) ２ ) ｏ - (8) ３ ) ｐ - (8) ４ ) ｑ - It has, During the ceremony, -L １ , L ２ , L ３ , and L ４ These are linkers that, individually and together, connect Z to D. -n, o, p, and q are each 0 or 1, where n + o + p + q = 1, 2, 3, or 4. (a) Linker L １ teeth, -(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 、 It is expressed as, in the formula, -d and d' are 0 or 1 respectively. -e and e' are integers in the range of 1 to 10, respectively. -f and f' are 0 or 1, respectively. -g and g' are integers in the range of 0 to 10, respectively; -k = 0 or 1, where k = 1, d = 0, -A is a sulfamide group according to structure (23), 【Chemistry 1】 In the formula, a = 0 or 1, R １３ is hydrogen, C １～ C ２４ alkyl group, C ３～ C ２４ Cycloalkyl groups, C ２～ C ２４ (hetero)aryl group, C ３～ C ２４ Alkyl (hetero)aryl group, and C ３～ C ２４ Selected from the group consisting of (hetero)arylalkyl groups, the C １～ C ２４ alkyl group, the C ３～ C ２４ Cycloalkyl group, the C ２～ C ２４ (hetero)aryl group, the C ３～ C ２４ Alkyl (hetero)aryl group, and the C ３～ C ２４ (Hetero)arylalkyls are optionally substituted with O, S, and NR １４ Optionally interrupted by one or more heteroatoms selected from R １４ These are, independently, hydrogen and C １～ C ４ Selected from the group consisting of alkyl groups, or R １３ This is D connected to N via the spacer portion. -W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH ２ ) ｍ C(O)-, -C(O)(CH ２ ) ｍ C(O)NH-, or -(4-Ph)CH ２ NHC(O)(CH ２ ) ｍ C(O)NH-, where m is an integer in the range of 0 to 10. -B is -CH ２ -CH ２ -O- or -O-CH ２ -CH ２ - is a part, or (B) ｅ is, -(CH ２ -CH ２ -O) ｅ１ -CH ２ -CH ２ - This is a part, and in the formula, e1 is an integer in the range of 1 to 10. -N ＊ is -(A) ｄ -(B) ｅ -(A) ｆ -(C(O)) ｇ’ Two examples of - are branched nitrogen atoms to which two (C(O)) are attached, and both (C(O)) ｇ’ moieties are -(L ２ ) ｏ -(L ３ ) ｐ -(L ４ ) ｑ is connected to -D, where L ２ , L ３ , L ４ , o, p, q, and D are each individually selected and / or (b) Linker L ２ It is a peptide spacer, and / or (c) Linker L ３ It is a self-sacrificing spacer, and / or (d) Linker L ４ is structure-NR ２２ - (C ｚ It is an aminoalkanoic acid spacer following the formula -alkylene)-C(O)-, where x is an integer in the range of 1 to 20, and R ２２ is H or C １ ~C ４ It is alkyl, or Linker L ４ is structure-NR ２２ - (CH ２ -CH ２ -O) ｅ６ - (CH ２ ) ｅ７ An ethylene glycol spacer according to -C(O)-, where 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 C １ ~C ４ It is alkyl, or Linker L ４ is structure-NR ２２ - (C ｚ -Alkilen)-NR ２２ - (C(O)) ｈ A diamine spacer that follows the formula, where h is 0 or 1, and x is an integer in the range of 1 to 10, R ２２ is H or C １ ~C ４ The antibody conjugate according to claim 1, wherein it is alkyl.

7. The antibody conjugate according to claim 1, wherein b = 1 and the conjugation is via the antibody glycan.

8. Z-L-D has a structure selected from the group consisting of (X) to (XII), 【Chemistry 4】 In the equation, the dashed line indicates a connection to Z, and L ２ , L ４ The antibody conjugate according to claim 1, wherein o and q are as defined in claim 6.

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

10. According to structure (XII) or (XIII), q = 0, o = 1, L ２ The antibody conjugate according to claim 8, wherein the spacer is a peptide spacer containing one to five amino acids, the antibody is hRS7 as defined in claim 5, and the payload is exatecan.

11. Z-L-D has the structure of (XIIIe), 【Chemistry 5-1】 The antibody conjugate according to claim 1, wherein the wavy line indicates a connection to Z, and the antibody is hRS7 as defined in claim 5.

12. A process for preparing the antibody conjugate described in claim 1, (i) An antibody containing the y-core N-acetylglucosamine (GlcNAc) moiety (y = 1, 2, 3, or 4) is subjected to the reaction of formula S(F) in the presence of a catalyst. ｘ -P (in the formula, S(F) ｘ is a sugar derivative containing x reactive groups F that can react with reactive group Q, where x is 1 or 2, and P is a monophosphate or diphosphate nucleoside, and the catalyst is the S(F) ｘ The compound (26) can be brought into contact with the core-GlcNAc portion, AB-[GlcNAc(Fuc) ｗ -S{F} ｘ ] ｙ （２６） During the ceremony, -AB is an antibody that can target Trop-2 expressing tumors. -Fuc is fucose, -w is 0 or 1) to obtain a modified antibody according to, (ii) The modified antibody, structure (2), Q-L-D （２） During the ceremony, -Q is the reactive part, -L is a linker that connects Z to D, -D is reacted with a compound according to exatecan, A process comprising obtaining the antibody conjugate according to structure (1).

13. The process according to claim 12, wherein the reaction in step (ii) is a nucleophilic reaction or cycloaddition.

14. The process according to claim 12, wherein Q is a click probe comprising a (hetero)cycloalkyne moiety or a (hetero)cycloalkene moiety.

15. An in vitro method comprising targeting Trop-2 expressing cells and contacting the antibody conjugate described in claim 1 with cells present in a target that may express Trop-2.

16. The method according to claim 15, wherein targeting the Top-2 expressing cells comprises one or more of the following: treatment, imaging, diagnosis, prevention, inhibition, and reduction of the proliferation of the Top-2 expressing cells.

17. The method according to claim 15 or 16, wherein the subject is suffering from oral cancer, pancreatic cancer, stomach cancer, ovarian cancer, colorectal cancer, breast cancer, or lung cancer.

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