Antibody conjugates for targeting tumors expressing PTK7

Abstract

The present invention relates to antibody conjugates which are particularly suitable for targeting cells expressing PTK7, in particular tumor cells. An antibody conjugate according to the present invention has structure (1): AB-[(L6) b-{Z-L-D} x] y (1) wherein AB is an antibody capable of targeting a tumor expressing PTK7; l is a joint connecting Z to D; z is a linking group; l6 is-GlcNAc (Fuc) w-(G) j-S-(L7) w '-wherein G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L7 is-N (H) C (O) CH2-,-N (H) C (O) CF2-or-CH2-; d is selected from the group consisting of an anthracycline, a camptothecin, a tubulysin, an endiyne, an amanitine, a duocarmycin, a maytansinoid, an aurestatin, eribulin, a BCL-XL inhibitor, a miscanthus semiaquilaria, a KSP inhibitor, a TLR agonist, an indolo-benzodiazepine dimer or a pyrrolo-benzodiazepine dimer (PBD), and an analog or prodrug thereof; b is 0 or 1; x is 1 or 2; and y is 1, 2, 3 or 4. The invention further relates to a method for preparing said antibody conjugate of structure (1) and to the use of said antibody conjugate of structure (1).

Description

Technical Field

[0001] The present invention belongs to the field of bioconjugates. More specifically, the present invention relates to antibody-drug conjugates for targeted therapy of patients with cancer, particularly tumors expressing PTK7. Background Art

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

[0003] Current ADCs are typically prepared by various conjugation techniques (summarized in Figure 1In the present invention, the conjugation technology is mainly based on the conjugation of maleimide to cysteine ​​side chains or activated esters to lysine side chains. To produce ADCs based on native cysteine ​​that naturally participates in disulfide bonds, the sulfhydryl groups in the side chains can be released by subjecting the antibody to a suitable reducing agent (such as TCEP or DTT) and then treating it with a maleimide-functionalized linker-drug. In the event that only a total of eight free sulfhydryl groups are not fully alkylated, the resulting ADC is generally composed of a mixture of positional isomers. Alternatively, to produce site-specific ADCs, antibodies can be generated by mutating one or more amino acids at defined positions in the sequence to cysteine, the side chain of which can be selectively released for alkylation via 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, and HC-442C. Additional cysteines can also be inserted into the sequence, such as HC-1239C. 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, specific non-natural (non-canonical) amino acids can also be designed into the amino acid sequence of the antibody to provide unique handles for chemical conjugation, such as ketones, acetylene, azide, cycloalkyne or cycloalkenes to react with oxime, azide, alkyne or tetrazine respectively. However, the disadvantage of the latter method is that the natural sequence of the antibody must be redesigned, which is time-consuming and expensive and may also lead to instability problems.

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

[0005] Chemoenzymatic conjugation of glycans (including first trimming the glycans) is known in the art and has been described by van Geel et al., Bioconjugate Chem. 2015, 26, 2233 and schematically depicted in Figure 2In brief, monoclonal antibodies are treated with endoglycosidases to trim the glycans to the core GlcNAc (directly attached to Asn-297), and then the azido-modified sugar is transferred under the action of glycosyltransferases. Various structures of UDP-azido sugars such as Figure 3 As shown. One particularly suitable combination involves the transfer of GalNAz 2b (2-azidoacetyl-N-galactosamine) under the action of a mutant galactosyltransferase GalT (Y289L), as disclosed in WO 2007 / 095506, EP 2911699 B1, and van Geel et al. Another useful combination requires the treatment of GlcNAz with α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT1) and α-1,6-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT2), and as disclosed in WO 2018 / 126092 and WO 2021 / 248048.

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

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

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

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

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

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

[0012] Protein tyrosine kinase 7 (PTK7) is a highly conserved member of the receptor tyrosine kinase pseudokinase family, with no observed kinase activity across species. Genetic and biochemical studies have demonstrated a critical role for PTK7 in non-canonical Wnt signaling, and PTK7-deficient embryos exhibit severe developmental defects in planar cell polarity. There is also evidence for additional functions for PTK7 in the vascular endothelial growth factor (VEGF), semaphorin / plexin, and canonical Wnt signaling pathways. The oncogenic function of PTK7 has been demonstrated in colon, lung, breast, ovarian, and esophageal cancers, and PTK7 promotes cell survival and chemotherapy resistance in acute myeloid leukemia.

[0013] Antibodies against PTK7 are known in the art, as disclosed in US9777070B2 (H23 and H24, also known as cofiluzumab), US9,777,070B2, US9,505,845B2 / US9,102,738B2 (4D6, 12C6, 12C6A / 1F12 and 7C8), and WO2015 / 168019 (e.g., Hu23 and Hu58).

[0014] ADCs targeting PTK7 are known in the art, such as PF-06647020 / ABBV-647 (cofiluzumab pertin), which is based on a humanized anti-PTK7 antibody and an auristatin microtubule inhibitor payload (Aur0101). PF-06647020 was shown to induce prolonged tumor regression in preclinical models of patient-derived tumor xenografts and to induce a tolerable safety profile and preliminary clinical activity in patients with previously treated locally advanced / metastatic PTK7-positive NSCLC, TNBC, and platinum-resistant OvCa when dosed every 2 or 3 weeks. Summary of the Invention

[0015] The inventors have developed antibody conjugates that are very suitable for targeting cells expressing PTK7, in particular tumors. Therefore, the antibody conjugates according to the present invention are very suitable for treating PTK7-positive cancers, in particular colon cancer, lung cancer, breast cancer, ovarian cancer and esophageal cancer.

[0016] In a first aspect, the present invention relates to an antibody conjugate. Related thereto, in a second aspect, the present invention relates to a method for preparing an antibody conjugate according to the present invention. In a third aspect, the present invention relates to a method for targeting cells expressing PTK7. Related thereto is a first medical use of the antibody conjugate according to the present invention, and a second medical use for treating cancer. Finally, the present invention relates to the use of a conjugation mode for increasing the therapeutic index of an antibody conjugate in the treatment of tumors expressing PTK7. DETAILED DESCRIPTION

[0017] definition

[0018] The verb "to comprise" and its conjugations as used in this description and claims are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0019] Furthermore, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. Thus, the indefinite article "a" or "an" generally means "at least one."

[0020] A linker is defined herein as a portion that connects (covalently links) two or more elements of a compound. A linker may include one or more spacer moieties. A spacer moiety is defined herein as a portion that separates (i.e., provides a distance between) two (or more) portions of a linker and covalently links them together. A linker can be, for example, a portion of a linker-construct, a linker-conjugate, a linker-payload (e.g., a linker-drug), or an antibody conjugate, as defined below.

[0021] A "hydrophilic group" or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that impart improved polarity to the molecule to which it is attached and thus provide improved water solubility. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonate groups, phosphate groups, acylsulfonamide groups, or carbamoylsulfonamide groups. In addition to higher solubility, other effects of the hydrophilic group include improved click conjugation efficiency and, once incorporated into the antibody-drug conjugate: reduced aggregation, improved pharmacokinetics, resulting in higher efficacy and in vivo tolerability.

[0022] The term "salt thereof" means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, but this is not necessary for salts that are not intended to be administered to a patient. For example, in a salt of a compound, the compound can be protonated by an inorganic or organic acid to form a cation, with a conjugate base of the inorganic or organic acid serving as the anionic component of the salt. The term "pharmaceutically acceptable salt" means a counterion that is acceptable for administration to a patient, such as a mammal (having acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable" salts refer to pharmaceutically acceptable salts of compounds, which are derived from various organic and inorganic counterions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

[0023] The term "enediyne" or "enediyne antibiotic" or "enediyne-containing cytotoxin" refers to any cytotoxin known in the art that is characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule, and includes neocarzinostatin (NCS), C-1027, kedarcidin (KED), maduropeptin (MDP), N1999A2, sporolide (SPO), cyanosporaside (CYA and CYN), and fijiolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamicin, shishijimicin, and uncialamycin (UCM).

[0024] The term "alkylamino sugar" as used herein refers to a tetrahydropyranyl moiety attached to an alcohol functionality via its 2-position, thereby forming an acetal functionality, and further substituted with (at least) one N-alkylamino group at position 3, 4 or 5. "N-alkylamino group" herein refers to an amino group having one methyl, ethyl or 2-propyl group.

[0025] The term "click probe" refers to a functional moiety capable of undergoing a click reaction, i.e., two compatible click probes undergo a click reaction with each other such that they are covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of the present invention, the click probe Q in the compound according to the invention is capable of reacting with the click probe F on the (modified) protein such that, when the click reaction occurs, a conjugate is formed in which the protein is conjugated to the compound according to the invention. Here, F and Q are compatible click probes.

[0026] "Acylsulfonamide moiety" is defined herein as a sulfonamide moiety (H2NSO2NH2) that is N-acylated or N-carbamylated at one end of the molecule and N-alkylated (single or double) at the other end of the molecule. In the context of the present invention, and particularly in the Examples, this group is also referred to as "HS".

[0027] A "coding sequence" or a sequence "encoding" an expression product, such as an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence may include a start codon (usually ATG) and a stop codon.

[0028] The term "gene" means a DNA sequence encoding or corresponding to a specific amino acid sequence, which comprises all or part of one or more proteins or enzymes, and which may or may not include a regulatory DNA sequence, such as a promoter sequence, which, for example, determines the conditions for gene expression. Some genes are not structural genes and can be transcribed into RNA from DNA, but are not translated into amino acid sequences. Other genes may serve as regulators of structural genes or regulators of DNA transcription. Specifically, the term gene can mean a genomic sequence encoding a protein, i.e., a sequence comprising regulators, promoters, introns, and exon sequences.

[0029] The term "glycoprotein" is used herein in its normal scientific sense and refers to a protein comprising one or more monosaccharide or oligosaccharide chains ("glycans") covalently bonded to the protein. Glycans can be attached to hydroxyl groups on the protein (O-linked glycans), such as those of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline, or to amide functional groups on the protein (N-glycoproteins), such as asparagine or arginine, or to carbons on the protein (C-glycoproteins), such as tryptophan. Glycoproteins can contain more than one type of glycan, can contain a combination of one or more monosaccharide and one or more oligosaccharide glycans, and can contain a combination of N-linked, O-linked, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and therefore qualify as glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0030] The term "glycan" is used herein with its normal scientific meaning and refers to a monosaccharide or oligosaccharide chain connected to a protein. Therefore, the term glycan refers to the carbohydrate portion of a glycoprotein. A glycan is connected to a protein via the C-1 carbon of a sugar, which may not be further substituted (monosaccharide), or may be further substituted (oligosaccharide) at one or more of its hydroxyl groups. Naturally occurring glycans typically contain 1 to about 10 sugar moieties. However, when a longer sugar chain is connected to a protein, the sugar chain is also considered to be a glycan in this article. The glycan of a glycoprotein can be a monosaccharide. Typically, the monosaccharide glycan of a glycoprotein is composed of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to the protein. A glycan can also be an oligosaccharide. The oligosaccharide chain of a glycoprotein can be straight or branched. In oligosaccharides, the sugar directly attached to the protein is called a core sugar. In oligosaccharides, the sugar that is not directly attached to the protein but is attached to at least two other sugars is called an inner sugar. In oligosaccharides, it is not directly connected to the protein but connected to a single other sugar, that is, the sugar that does not carry other sugar substituents on one or more of its other hydroxyl groups is referred to as a terminal sugar. For the avoidance of doubt, multiple terminal sugars can be present in the oligosaccharides of glycoproteins, but there is only one core sugar. Polysaccharides can be O-linked polysaccharides, N-linked polysaccharides or C-linked polysaccharides. In O-linked polysaccharides, monosaccharides or oligosaccharide polysaccharides are typically bonded to the O atoms in the amino acids of proteins via the hydroxyl groups of serine (Ser) or threonine (Thr). In N-linked polysaccharides, monosaccharides or oligosaccharide polysaccharides are typically bonded to proteins via the amide nitrogen in the side chains of asparagine (Asn) or arginine (Arg). In C-linked polysaccharides, monosaccharides or oligosaccharide polysaccharides are bonded to the C atoms in the amino acids of proteins, typically to the C atoms of tryptophan (Trp).

[0031] The term "antibody" (AB) is used in this article with its normal scientific meaning.Antibody is a protein produced by the immune system that can recognize and bind to a specific antigen.Antibody is an example of a glycoprotein.The term antibody herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies.The term "antibody" is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens.The term "antibody" is intended to include complete antibodies, but also includes antibody fragments, such as antibody Fab fragments, F (ab') 2, Fv fragments or Fc fragments, scFv-Fc fragments, mini antibodies, double antibodies, or scFv from cleaved antibodies. In addition, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0032] Antibodies can be natural or conventional antibodies, 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: λ (l) and κ (k). The light chain includes two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, one variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity for the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and is composed of the variable portion of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass or allotype (e.g., human G1m1, G1m2, 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) of immunoglobulin molecule. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2 or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. The antibody can be engineered in the Fc domain to enhance or eliminate binding to Fc-γ 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 Fc-γ receptors can also be achieved by mutating the N297 amino acid to any amino acid other than asparagine, by mutating the T299 amino acid to any amino acid other than threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies using, for example, PNGase F or endoglycosidases. Immunoglobulins can be derived from any species, including human, mouse, or rabbit sources. Each chain contains different sequence domains.

[0033] The percentage of "sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). A sequence that is "at least 85% identical to a reference sequence" is one that has 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its entire length to the entire length of the reference sequence.

[0034] The term "CDR" refers to complementarity determining region: the specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues primarily from the hypervariable regions or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and, therefore, the binding site. The complementarity determining region or CDR therefore refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site includes six CDRs, which comprise a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0035] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule that is directed against a single amino acid sequence of a specific antigen, and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0036] The term "chimeric antibody" refers to an engineered antibody that comprises one or more regions from an antibody and one or more regions from one or more other antibodies in its broadest sense. In one embodiment, a chimeric antibody comprises a VH domain and a VL domain derived from an antibody of a non-human animal, which are combined with a CH domain and a CL domain of another antibody (in one embodiment, a human antibody). As non-human animals, any animal such as mice, rats, hamsters, rabbits, etc. can be used. A chimeric antibody can also refer to a multispecific antibody that is specific for at least two different antigens.

[0037] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and, for example, has been modified to replace certain amino acids in the framework regions of the VH and VL domains in order to avoid or minimize an immune response in humans. The constant domains of humanized antibodies are most often human CH and CL domains. A "fragment" of a (conventional) antibody comprises a portion of a complete antibody, particularly the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody can also be a single domain antibody, such as a heavy chain antibody or VHH.

[0038] As used herein, "PTK7," also known as colon cancer kinase 4 (CCK4), refers to a highly conserved member of the pseudokinase family of receptor tyrosine kinases that has no observable kinase activity in different species. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A representative set of reactive groups (F) are shown, which, when present in a biomolecule, will form linker groups Z (1a-1h) upon reaction with reactive group Q. Functional group F may be naturally occurring or artificially introduced (engineered) into a biomolecule at any chosen position.

[0040] Figure 2 Schematic diagram of how to obtain antibody conjugates from any monoclonal antibody by a two-stage process. In the first stage, azide-modified UPD-Gal or UDP-GalNAc can be attached to a monoclonal antibody in a one-pot process involving (a) trimming of the glycan (to core GlcNAc) by an endoglycosidase and (b) attaching the azide-sugar under the action of a glycosyltransferase (galactosyltransferase or its mutant or GalNAc-transferase), thereby generating a β-glycosidic 1-4 bond between the azide-modified GalNAc and GlcNAc. In the second stage, the azide-modified antibody is reacted with an appropriately functionalized cyclooctyne to produce the antibody conjugate.

[0041] Figure 3 Several structures of UDP sugar derivatives of galactosamine are shown, which can be modified at the 2-position with, for example, a 3-mercaptopropionyl group (2a), an azidoacetyl group (2b), or an azidodifluoroacetyl group (2c), or at the 6-position of N-acetylgalactosamine (2d) with an azido group.

[0042] Figure 4 Cyclooctyne (AT), suitable for metal-free click chemistry, is shown to be the first choice for the reactive moiety Q.

[0043] Figure 5 A set of exemplary toxic payloads for conjugation to various monoclonal antibodies targeting PTK7 according to the present invention is shown. The point of attachment of the linker (to the amino group present in the payload) is indicated by an arrow. Preferred conjugates according to the present invention contain a payload including a point of attachment, such as Figure 5 shown.

[0044] Figure 6 Shown are the structures of various camptothecins, which are preferred payloads in the context of the present invention.

[0045] Figure 7 Depicted is the structure of a BCN-linker drug for the preparation of ADCs via click chemistry of azidosugar remodeled antibodies with the following payloads (3 = ixotecan, 4 = MMAE, 5a = calicheamicin gamma 1 I , 5b = glycine-calicheamicin γ1 I , 6 = 6-aminohexanoyl-maytansinoid).

[0046] Figure 8 A shows the binding of cofilotuzumab to hPTK7 and cPTK7, corrected for background. Figure 8 B shows the binding of all cofiluzumab-based ADCs to hPTK7, which is comparable to the corresponding mAbs.

[0047] Figure 9 In vitro efficacy data for A431 cells treated with increasing concentrations of ADCs cofetiuzumab-4, cofetiuzumab-5b, and negative control B12-3 are shown. Both cofetiuzumab ADCs showed cell killing, with IC 50 The values ​​were 3.9 nM and 0.1 nM, respectively.

[0048] Figure 10 A and 10C show in vivo efficacy data over time on the NCI-H446 tumor model expressing PTK7 ( Figure 10 A: until t = 14 days; Figure 10C : until t = 44 days). Cofetiuzumab-3 resulted in tumor regression with a clear dose response (2.59, 5.18, and 10.36 mg / kg). Administration of the cofetiuzumab-5b ADC also showed a dose response leading to tumor growth delay (2.59 and 5.18 mg / kg). Other ADCs, including cofetiuzumab-Aur0101, showed only minor tumor growth delay (similar to the isotype control ADC B12-3). Figure 10 B and 10D show the changes in the body weight of mice over time ( Figure 10 B: until t = 14 days; Figure 10D: until t = 28 or 44 days).

[0049] Figure 11 Binding of 4D5-3, 12C6a-3, 12C6-3, and 7C8-3 to hPTK7, cPTK7, and rPTK7 is shown. 12C6-3 exhibited the highest relative binding affinity to human and cynomolgus monkey PTK7. 12C6a-3 did not exhibit any binding at all. 4D5-3 and 7C8-3 exhibited similar binding to hPTK7 and cPTK7, whereas 7C8 clearly had a higher binding affinity to rPTK7.

[0050] Figure 12A In vivo efficacy data over time are shown in the PTK7-expressing NCI-H446 tumor model. 12C6-3 showed no tumor growth inhibition, similar to vehicle. 7C8-3 and 4D5-3 both showed minimal tumor growth delay. Cofetiuzumab-3 showed tumor growth inhibition but also early tumor regrowth. 12C6-3 demonstrated 99.7% tumor growth inhibition through day 40. Figure 12B Shown are the changes in the body weight of mice over time. Figure 12C Survival over time in Kaplan-Meier plots is shown for the NCI-H446 model.

[0051] Figure 13A In vivo efficacy data over time in the PTK7-expressing BR1282 PDX tumor model is shown. 12C6-3 exhibited superior tumor growth inhibition compared to all 7C8-3, 4D5-3, and 12C6a-3, and even compared to cofetizumab-Aur0101. When compared to cofetizumab-3, both ADCs demonstrated similar responses and equally good tumor growth inhibition. Figure 13B Shown are the changes in the body weight of mice over time.

[0052] Figure 14A In vivo efficacy data over time are shown in the PTK7-expressing NCI-H446 tumor model. Olaparib did not show a significant effect as a single treatment. Both 12C6-3 and 12C6-9 showed tumor growth inhibition / arrest. However, the combination of Olaparib and 12C6-3 showed a clear additive effect, resulting in complete responses in 6 of 8 mice at day 21. This study is still ongoing. Figure 14B Shown are the changes in the body weight of mice over time.

[0053] Figure 15The in vivo tolerability data for 12C6-3 in female Sprague Dawley rats over time are presented. No toxicity or weight loss was observed at any concentration. Therefore, the maximum tolerated dose is greater than 150 mg / kg.

[0054] The present invention

[0055] In a first aspect, the present invention relates to an antibody conjugate having the general structure (1),

[0056] AB–[(L 6 ) b –{Z–L–D} x ] y (1)

[0058] in:

[0059] -AB is an antibody that can target tumors expressing PTK7;

[0060] -L is the linker connecting Z to D;

[0061] -Z is a linking group;

[0062] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0063] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0064] -b is 0 or 1;

[0065] -x is 1 or 2; and

[0066] -y is 1, 2, 3, or 4.

[0067] The present invention also encompasses salts, preferably pharmaceutically acceptable salts, of the antibody conjugate according to structure (1).

[0068] In a second aspect, the present invention relates to a method for preparing an antibody conjugate according to the present invention, the method comprising reacting a compound according to the general structure (2) with an antibody (3). The compound according to the general structure (2) comprises a reactive moiety Q and an antibody reactive moiety F, the antibody reactive moiety F being capable of reacting with Q in a conjugation reaction, wherein Q and F react to form a linker Z. In this reaction, a conjugate according to the general structure (1) is formed. The method according to this aspect involves the following bioconjugation reaction:

[0069]

[0070] Below, the antibody conjugate according to structure (1) is first defined. The structural features of the antibody conjugate according to structure (1) also apply to the compound according to structure (2) and the antibody according to structure (3), because except for the reactive moieties F and Q, those are not changed in the conjugation reaction, which are converted to the linker Z when the compound according to structure (2) reacts with the antibody according to structure (3).

[0071] In a third aspect, the present invention relates to the use of an antibody conjugate according to structure (1) for targeting cells expressing PTK7. 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).

[0072] As will be understood by those skilled in the art, the definitions of chemical moieties and their preferred embodiments apply to all aspects of the invention.

[0073] General structure of antibody conjugates (1)

[0074] In a first aspect, the present invention relates to an antibody conjugate having the general structure (1):

[0075] AB–[(L 6 ) b –{Z–L–D} x ] y (1)

[0077] in:

[0078] -AB is an antibody that can target tumors expressing PTK7;

[0079] -b is 0 or 1;

[0080] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0081] -Z is a linking group;

[0082] -L is the linker connecting Z to D;

[0083] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0084] -x is 1 or 2; and

[0085] -y is 1, 2, 3, or 4.

[0086] Antibody AB

[0087] The antibody conjugates according to the present invention contain antibodies capable of targeting cells expressing PTK7, particularly tumor cells. PTK7 is a known target for cancer therapy. The term "expression" is used as commonly used in the art and refers to overexpression of the target relative to expression in healthy tissue. Antibodies capable of targeting tumors expressing PTK7 may also be referred to as anti-PTK7 antibodies, PTK7-targeting antibodies, or PTK7-binding antibodies. Anti-PTK7 antibodies selectively bind to cells expressing PTK7. Anti-PTK7 antibodies are known in the art, and any suitable antibody may be used in the context of the present invention.

[0088] The Fc region of these antibodies may have one or more mutations, such as 0 to 10 mutations or 0 to 5 mutations. Particularly preferred are mutations that alter binding to the FcRn receptor to regulate the half-life of the antibody. For example, the inclusion of mutations Met to Tyr, Ser to Thr, and Thr to Glu in the IgG1 Fc heavy chain amino acid 254–260 region (commonly referred to as YTE) results in approximately 11 times higher binding of the antibody to human FcRn, thereby extending the circulating half-life by about 3.5 times. For example, the YTE mutations of cofilotuzumab are Met254Tyr, Ser256Thr, and Thr258Glu. Thus, in one embodiment, the antibody has a half-life of less than 2.5x 10-6 M, preferably 0.05 to 0.99 x 10 -6 M, more preferably in the range of 0.1 to 0.49 x 10 -6 M, most preferably in the range of 0.2 to 0.4 x 10 -6 Apparent human FcRn binding affinity K in the range of M D，表观 Apparent binding affinity K D，表观 The assay can be performed according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, the antibody AB is a YTE mutant of a preferred antibody as defined above or below.

[0089] In a preferred embodiment, the antibody is cofeluzumab. Cofeluzumab can also be defined as comprising a light chain sequence according to SEQ ID No. 8 and a heavy chain sequence according to SEQ ID No. 7, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0090] In a preferred embodiment, the antibody is 12C6. 12C6 can also be defined as comprising a light chain sequence according to SEQ ID No. 20 and a heavy chain sequence according to SEQ ID No. 19, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0091] In a preferred embodiment, the antibody is 12C6a. 12C6a can also be defined as comprising a light chain sequence according to SEQ ID No. 22 and a heavy chain sequence according to SEQ ID No. 19, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0092] In a preferred embodiment, the antibody is 4D5. 4D5 can also be defined as comprising a light chain sequence according to SEQ ID No. 16 and a heavy chain sequence according to SEQ ID No. 15, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0093] In a preferred embodiment, the antibody is 7C8. 7C8 can also be defined as comprising a light chain sequence according to SEQ ID No. 25 and a heavy chain sequence according to SEQ ID No. 26, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0094] In a preferred embodiment, the antibody is Hu23. Hu23 can also be defined as comprising a light chain sequence according to SEQ ID No. 33 and a heavy chain sequence according to SEQ ID No. 31, 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, exitecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exitecan, and most preferably with exitecan as the payload D.

[0095] In a preferred embodiment, the antibody is Hu58. Hu58 can also be defined as comprising a light chain sequence according to SEQ ID No. 37 and a heavy chain sequence according to SEQ ID No. 35, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exotecan, and most preferably with exotecan as the payload D.

[0096] Thus, in preferred embodiments, the antibody is selected from cofilizumab, 12C6, 12C6a, 4D5, 7C8, Hu23, and Hu58, all as defined above, more preferably selected from cofilizumab, 12C6, 4D5, and 7C8, even more preferably selected from cofilizumab and 12C6. In one embodiment, the antibody is cofilizumab. In one embodiment, the antibody is 12C6.

[0097] Preferred antibodies are those whose V L and V H The variable domains are defined as follows, which together form the variable domain that binds the antigen. Thus, in a preferred embodiment, the antibody comprises: a V domain selected from the group consisting of SEQ ID No. 2, 6, 9, 11, 14, 18, 21, 24, 32 and 36. L domain, and a V selected from the group consisting of SEQ ID No. 1, 5, 10, 12, 13, 17, 23, 30 and 34 H domain, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a particularly preferred embodiment, the antibody comprises V of SEQ ID No. 6 Ldomain and V of SEQ ID No.5 H Domains wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.

[0098] The above sequence identity refers to V L and V H The complete sequence of the structural domains. Although some variation in the sequence is tolerated throughout the sequence of these domains without impairing binding to PTK7, it is preferred that the complementarity determining regions (CDRs) have a high degree of sequence identity to ensure that binding to PTK7 is not significantly impaired. The positions of the CDRs are given in the table below. Thus, it is preferred that the antibody contain: V selected from the group consisting of SEQ ID No. 2, 6, 9, 11, 14, 18, 21, 24, 32 and 36 L domain, and a V selected from the group consisting of SEQ ID No. 1, 5, 10, 12, 13, 17, 23, 30 and 34 H domain, preferably V of SEQ ID No.6 L domain and V of SEQ ID No.5 H domains, wherein the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. It is understood by those skilled in the art that the V L and V H The domains can be combined with appropriate constant domains to form complete antibodies.

[0099] Preferred V L domain

[0100] SEQ ID No. CDR (amino acid number within the sequence) 2 23-34|50-56|89-97 6 24-38|54-60|93-101 9 24-39|55-61|94-102 11 24-34|50-56|89-97 14 24-34|50-56|89-97 18 24-25|51-57|90-99 21 24-34|50-56|89-97 24 24-34|50-56|89-98

[0101] Preferred V H domain

[0102]

[0103]

[0104] In a particularly preferred embodiment, the antibody comprises V of SEQ ID No. 2 L domain and V of SEQ ID No.1 H domain, or V of SEQ ID No.6 L domain and V of SEQ ID No.5 H domain, or V of SEQ ID No.9 L domain and V of SEQ ID No.10H domain, or V of SEQ ID No.11 L domain and V of SEQ ID No.12 H domain, or V of SEQ ID No. 14 L domain and V of SEQ ID No.13 H domain, or V domain of SEQ ID No. 18 or 21 L domain and V of SEQ ID No.17 H domain, or V of SEQ ID No.24 L domain and V of SEQ ID No.23 H domain, or V of SEQ ID No. 32 L domain and V of SEQ ID No.30 H domain, or V of SEQ ID No.36 L domain and V of SEQ ID No.34 H Herein, the sequence identity defined above for the complete sequence as well as for the CDRs applies.

[0105] Alternatively, the antibody is defined by its light chain and heavy chain, which together form the antibody. Thus, in a preferred embodiment, the antibody comprises a light chain selected from the group consisting of SEQ ID Nos. 4, 8, 16, 20, 22, 26, 33 and 37, and a heavy chain selected from the group consisting of SEQ ID Nos. 3, 7, 15, 19, 25, 31 and 35, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a further preferred embodiment, the antibody comprises a light chain of SEQ ID No. 8 and a heavy chain of SEQ ID No. 7, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.

[0106] The above sequence identities refer to the complete sequences of the light and heavy chains. While some variation in sequence is tolerated throughout the entire sequence of these chains without impairing binding to PTK7, it is preferred that the CDRs have a high degree of sequence identity to ensure that binding to PTK7 is not significantly impaired. The following table shows the positions of the CDRs. Thus, it is preferred that the antibody comprises: a light chain selected from the group consisting of SEQ ID Nos. 4, 8, 16, 20, 22, 26, 33, and 37, and a heavy chain selected from the group consisting of SEQ ID Nos. 3, 7, 15, 19, 25, 31, and 35, wherein the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

[0107] Preferred light chain

[0108]

[0109]

[0110] Preferred heavy chain

[0111] SEQ ID No. CDR (amino acid number within the sequence) 3 26-37|52-67|100-109 7 26-35|50-65|97-108 15 31-35|50-66|99-104 19 31-35|50-65|98-101 25 31-35|50-65|99-115

[0112] In particularly preferred embodiments, the antibody comprises a light chain of SEQ ID No. 4 and a heavy chain of SEQ ID No. 3, or a light chain of SEQ ID No. 8 and a heavy chain of SEQ ID No. 7, or a light chain of SEQ ID No. 16 and a heavy chain of SEQ ID No. 15, or a light chain of SEQ ID No. 20 or 22 and a heavy chain of SEQ ID No. 19, or a light chain of SEQ ID No. 26 and a heavy chain of SEQ ID No. 25, or a light chain of SEQ ID No. 33 and a heavy chain of SEQ ID No. 31, or a light chain of SEQ ID No. 37 and a heavy chain of SEQ ID No. 35. Herein, the sequence identity defined above for the complete sequence as well as for the CDRs applies.

[0113] Connector L 6

[0114] If the reactive group F is directly attached to the antibody, or even part of the antibody structure, there is no linker L connecting AB to F (for the antibody of structure (3)) or to Z (for the conjugate of structure (1)). 6 and b = 0. This is the case, for example, for cysteine ​​conjugation and lysine conjugation. Alternatively, a linker L may be used. 6 A reactive group F is introduced onto the antibody, and a linker connects AB to F (for the antibody of structure (3)) or to Z (for the conjugate of structure (1)), in which case L is present. 6And b=1. If there is L 6 , then a reactive group F is typically introduced at the glycan of the antibody. This is the case, for example, via artificially introduced reactive group F conjugation, such as using transglutaminase or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyl-transferase). For example, a modified sugar residue S(F) can be introduced at the glycan x , extending the glycan with one monosaccharide residue S, which introduces x reactive groups F on the glycan of the antibody. In the most preferred embodiment, the conjugation occurs via the glycan of the antibody and b = 1. The conjugation site is preferably located at the heavy chain of the antibody.

[0115] If present, L 6 It is the linker that connects AB to F or Z and consists of –GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’ - represents, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 It is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–. Usually, L 6 At least in part, it is formed by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at or near the asparagine residue at position 297 of the heavy chain, which is modified by a complex type of glycan. It is preferred to use the naturally occurring glycosylation site of the antibody, but other glycosylation sites, including artificially introduced glycosylation sites, can also be used in the linker L. 6 Therefore, in a preferred embodiment, L 6 An amino acid located within the range of 250 to 350 of the heavy chain, preferably within the range of 280 to 310 of the heavy chain, more preferably within the range of 295 to 300 of the heavy chain, most preferably at position 297 of the heavy chain is linked to the antibody.

[0116] L 6 -GlcNAc(Fuc) w –(G) j – is a glycan of an antibody or a portion thereof. Thus, –GlcNAc(Fuc) w –(G) j– Typically derived from the original antibody, where GlcNAc is an N-acetylglucosamine moiety and Fuc is a fucose moiety. Fuc is typically bound to GlcNAc via an α-1,6-glycosidic bond. Typically, an antibody may (w=1) or may not (w=0) be fucosylated. In the context of the present invention, the presence of a fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue may also be referred to as a core-GlcNAc residue and is a monosaccharide directly attached to the peptide portion of the antibody.

[0117] S can be directly linked to the core-GlcNAc(Fuc) w part, i.e. j = 0, which means that before S attachment, the remaining part of the glycan is separated from the core-GlcNAc(Fuc) w This type of glycan trimming is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, in the core-GlcNAc(Fuc) w There are one or more monosaccharide residues between moiety and S, i.e. j is an integer in the range of 1 to 10, preferably j=2 to 5. In a preferred embodiment, (G) j is an oligosaccharide moiety comprising j monosaccharide residues G, wherein j is an integer ranging from 2 to 5. (G) j Typically linked to GlcNAc(Fuc) via a β-1,4 linkage w In a preferred embodiment, j is 3, 4 or 5. Although any monosaccharide that may be present in the polysaccharide can be used as G, each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose and N-acetylneuraminic acid. More preferably, G is selected from galactose, N-acetylglucosamine, mannose. The inventors have found that antibody conjugates with j below 4 do not show or hardly any binding to Fc-γ receptors, while antibody conjugates with j in the range of 4 to 10 do bind to Fc-γ receptors. Therefore, by selecting a certain value for j, the desired degree of binding to Fc-γ receptors can be obtained. Therefore, preferably j = 0, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably j = 0, 3, 4 or 5, most preferably the antibody is trimmed and j = 0.

[0118] S is a sugar or a sugar derivative. The term "sugar derivative" is used herein to represent a derivative of a monosaccharide, i.e. a monosaccharide comprising 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 (GlcNH ), galactosamine (GalNH ), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (also known as 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.

[0119] x is an integer representing the number of linking groups Z (for conjugate (1)) or reactive groups F (for antibody (3)) attached to the saccharide (derivative) S. Thus, the antibody according to the present invention contains a moiety S comprising x reactive moieties F. Each of these reactive moieties F reacts with the reactive moiety Q of the compound according to the general structure (2), such that x linking groups Z are formed and x compounds according to the general structure (2) are attached to a single occurrence of S. x is 1 or 2, preferably x=1.

[0120] The linker group Z (for conjugate (1)) or the reactive group F (for antibody (3)) can be directly linked to S, or a linker L can be present between S and Z or F. 7 . L 7 It is a linker connecting S and Z. It can be present (w'=1 or 2) or absent (w'=0) 7 Typically, each moiety Z can be linked to a 7 Connected to S, so in one embodiment x has w'=0. Preferably, there is no L 7 And each connecting part Z is directly connected to S. If present, L 7 It can be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. In a preferred embodiment, x=1 and w'=0 or 1, most preferably x=1 and w'=0.

[0121] y is an integer representing the number of sugar (derivative) S, each sugar (derivative) S having x reactive groups F or connected to x linking groups Z, said linking groups Z being connected to the antibody. y is 1, 2, 3 or 4, preferably y=2 or 4, most preferably y=2. Thus, the antibody contains y parts S, each of which contains x reactive parts F. Each of these reactive parts F reacts with the reactive part Q of the compound according to the general structure (2) so that x+y linking groups Z are formed, and x+y compounds according to the general structure (2) are connected to a single antibody. For example, due to the branched nitrogen atom N* in L, each compound according to the general structure (2) can contain multiple payloads. It is preferred that each compound according to the general structure (2) contains 1 or 2 occurrences of D, most preferably 2 occurrences of D. In a particularly preferred embodiment, the linker L 1 Contains a second occurrence of a D-attached branch nitrogen atom N*.

[0122] The amount of payload (D) molecules connected to a single antibody is referred to in the art as DAR (drug-antibody ratio). In the context of the present invention, preferably DAR is an integer in the range of 1 to 8, more preferably 2 or 4, most preferably DAR=4. In other words, DAR is preferably an integer in the range of (x+y) to [(x+y)×2], most preferably DAR=[(x+y)×2]. When the preferred value of x is 1 and the preferred value of y is 2, DAR is preferably 4. It should be understood that these are theoretical DAR values, and in practice, DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as random mixtures of antibody-drug conjugates, with DAR values ​​varying between each conjugate, and depending on the conjugation technology used, DAR may have a wide distribution (e.g., DAR=0 to 10) or a narrow distribution (e.g., DAR=3 to 4). In the case of such mixtures, DAR generally refers to the average DAR of the mixture. This is well known in the field of bioconjugation. However, in cases where conjugation occurs via the glycan (ie b = 1 and L is present 6 ), the antibody conjugates according to the present invention have a DAR close to the theoretical one. For example, when the theoretical DAR is 4, it is easy to obtain a DAR value higher than 3.6 or even higher than 3.8, which indicates that most of the antibodies in the reaction mixture have been fully reacted and the DAR is 4.

[0123] Linking group Z

[0124] Z is a linking group that covalently links the two parts of the conjugate according to the present invention. The term "linking group" herein refers to a structural element that is generated by the reaction between Q and F and that connects one part of the conjugate to another part of the same conjugate. As will be understood by those skilled in the art, the nature of the linking group depends on the type of reaction that results in the connection between the parts of the compound. For example, when the carboxyl group of RC(O)-OH reacts with the amino group of H2N-R' to form RC(O)-N(H)-R', R is connected to R' via the linking group Z and Z can be represented by the group -C(O)-N(H)-. Since the linking group Z is derived from the reaction between Q and F, it can take any form.

[0125] Since more than one reactive portion F can be present or introduced into an antibody, the antibody conjugate according to the present invention can contain more than one payload D per biomolecule, for example 1 to 8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D. Taking into account the symmetrical nature of antibodies, the number of payloads is usually an even number. In other words, when one side of the antibody is functionalized with F, the symmetrical counterpart will also be functionalized. Alternatively, if the naturally occurring sulfhydryl group of a cysteine ​​residue of a protein is used as F, the value of m can be any value and can vary between individual conjugates.

[0126] In the compound according to structure (1), the linker Z is optionally connected via a linker L. 6 D is connected to AB. Many reactions for connecting the reactive group Q to the reactive group F are known in the art. Therefore, a variety of linking groups Z may be present in the conjugate according to the present invention. In one embodiment, the reactive group Q is selected from the above options, preferably as Figure 1 As shown, the complementary reactive groups F and the linking groups Z obtained therefrom are known to those skilled in the art. Figure 1 It is shown that when a linker-conjugate comprising Q is conjugated to a biomolecule comprising a complementary reactive group F, F and Q as well as the linking group Z that will be present in the bioconjugate 3 Several examples of suitable combinations are given below.

[0127] For example, when F comprises or is a thiol group, the complementary group Q comprises an N-maleimido group and an alkenyl group, and the corresponding linking group Z is such as Figure 1 When F comprises or is a thiol group, the complementary group Q further comprises an amalonamide group.

[0128] For example, when F comprises or is an amino group, the complementary group Q includes a keto group and an activated ester group, and the corresponding linking group Z is such as Figure 1 shown.

[0129] For example, when F comprises or is a keto group, the complementary group Q includes an (O-alkyl)hydroxyamino group and a hydrazine group, and the corresponding linking group Z is such as Figure 1 shown.

[0130] For example, when F comprises or is an alkynyl group, the complementary group Q comprises an azido group, and the corresponding linking group Z is such as Figure 1 shown.

[0131] For example, when F comprises or is an azido group, the complementary group Q comprises an alkynyl group, and the corresponding linking group Z is such as Figure 1 shown.

[0132] For example, when F comprises or is a cyclopropenyl group, a trans-cyclooctenyl group or a cyclooctyne group, the complementary group Q comprises a tetrazinyl group, and the corresponding linking group Z is such as Figure 1 In these specific cases, Z is only an intermediate structure, and N2 will be eliminated to produce a dihydropyridazine (from the reaction with the olefin) or a pyridazine (from the reaction with the alkyne).

[0133] Further suitable combinations of F and Q and the properties of the resulting linker Z3 are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), in particular Chapter 3, pages 229-258, which are incorporated by reference. A list of complementary reactive groups suitable for bioconjugation methods is disclosed in Table 3.1 of Chapter 3, pages 230-232 of GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), and the contents of this table are expressly incorporated herein by reference.

[0134] In a preferred embodiment, the linker Z is obtained by a cycloaddition or nucleophilic reaction, preferably wherein the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition, or the nucleophilic reaction is a Michael addition or a nucleophilic substitution. This cycloaddition or nucleophilic reaction occurs via the reactive group F linked to S and the reactive group Q linked to D via L. Conjugation reactions via cycloaddition or nucleophilic reactions are known to those skilled in the art, and those skilled in the art are able to select appropriate reaction partners F and Q and will understand the properties of the resulting linker Z.

[0135] In a first preferred embodiment, Z is formed by a cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reactions) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are inverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and those skilled in the art know how to perform them.

[0136] Preferably, Z contains a moiety selected from the group consisting of a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide or imide group. The triazole moiety is particularly preferably present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e., is formed by Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e., is formed by Q comprising a (hetero)cycloalkene moiety. In a preferred embodiment, Z has the structure (Z1):

[0137]

[0138] Here, it is represented as ––– The bond is either a single bond or a double bond. In addition:

[0139] - Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za)-(Zj) defined below, wherein the carbon atom marked with ** corresponds to the carbon atom of (Z1) fused to ring Z represented by ––– The bond between the two carbon atoms;

[0140] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0141] –C 24 a group consisting of (hetero)arylalkyl groups;

[0142] -Y 2 It 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 (-) Balanced cationic sulfur atoms, where B (-) is an anion, and each R 31 R alone 15 or a connection to D, which is connected via L;

[0143] -u is 0, 1, 2, 3, 4, or 5;

[0144] - u' is 0, 1, 2, 3, 4 or 5, wherein u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0145] -v=an integer in the range of 8 to 16;

[0146] - Ring A is formed by cycloaddition and is preferably selected from (Za)-(Zj).

[0147] If depicted as ––– If the bond of is a double bond, then preferably u+u'=4, 5, 6, 7 or 8. Preferably, the wavy bond marked with * is connected to S, and the wavy bond marked with ** is connected to L.

[0148] Particularly preferably, Z comprises a (hetero)cycloalkene moiety, i.e. is depicted as ––– The bond is a double bond. In a preferred embodiment, Z is selected from the structures (Z2)-(Z20), as shown below:

[0149]

[0150]

[0151] Here, the connection to L is depicted by a wavy bond. B (–)It is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za)-(Zm) as depicted below, wherein the carbon atoms marked with ** correspond to two carbon atoms of the (hetero)cycloalkane ring (Z2)-(Z20) to which ring Z is fused. Preferred ring Z is selected from (Za)-(Zj), more preferably selected from (Za), (Zd) and (Zh), and most preferably ring Z has structure (Za). Since in the context of the present embodiment the linking group Z is formed by reaction with a (hetero)cycloalkyne, the above depiction is ––– The bond is a double bond.

[0152]

[0153]

[0154] In a further preferred embodiment, Z is selected from structures (Z21)-(Z38) and (Z38a), as shown below:

[0155]

[0156] Here, the connection to L is depicted by a wavy bond. In structure (Z38), B (–) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) to (Zm) as defined above, preferably from structures (Za) to (Zj).

[0157] In preferred embodiments, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to Structure (Z8), (Z26), (Z27), (Z28) or (Z37) or (Z38a), more preferably according to Structure (Z8), (Z26), (Z27), (Z28) or (Z37), which is optionally substituted. Each of these preferred options for Z is further defined below.

[0158] Thus, in a preferred embodiment, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Z37) is unsubstituted.

[0159] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z is structure (Z42) as further defined below.

[0160] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27) or (Z28), which is optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 independently selected from hydrogen, linear or branched C1-C 12 Alkyl group or C4-C 12 The aromatic ring in (Z40) is optionally O-sulfonylated at one or more positions, while the ring in (Z41) may 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 structure (Z43) as further defined below.

[0161] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37).

[0162]

[0163] In particularly preferred embodiments, Z comprises a cyclooctenyl group and is according to structure (Z42):

[0164]

[0165] In this article:

[0166] - The bond marked with * is connected to S, and the wavy bond marked with ** is connected to L;

[0167] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3(-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–

[0168] C 24 a group consisting of (hetero)arylalkyl groups;

[0169] -R 18 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 a group consisting of (hetero)arylalkyl groups;

[0170] -R 19 Selected from hydrogen, halogen, C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, said alkyl being optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Z (or Q) or D linked via a spacer moiety; and

[0171] -l is an integer in the range 0 to 10.

[0172] In preferred embodiments of groups according to structure (Z42), R 15 are independently selected from hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In preferred embodiments of groups according to structure (Z42), R 18 Independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably two R 18 are all H. In preferred embodiments of groups according to structure (Z42), R 19 is H. In preferred embodiments of the group according to structure (Z42), l is 0 or 1, more preferably l is 1.

[0173] In particularly preferred embodiments, Z comprises a (hetero)cyclooctynyl group and is according to structure (Z43):

[0174]

[0175] In this article:

[0176] - The bond marked with * is connected to S, and the wavy bond marked with ** is connected to L;

[0177] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0178] –C 24 a group consisting of (hetero)arylalkyl groups;

[0179] -Y is N or CR 15 .

[0180] In preferred embodiments of groups according to structure (Z43), R 15 are independently selected from hydrogen, halogen, -OR16 、-S(O)3 (-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from hydrogen and -S(O)3 (-) In preferred embodiments of the group according to structure (Z43), Y is N or CH, more preferably Y═N.

[0181] In particularly preferred embodiments, Z comprises a heterocycloheptynyl group and is according to structure (Z37) or (Z38a), preferably according to structure (Z37) wherein ring Z is a triazole:

[0182]

[0183] In an alternative preferred embodiment, Z comprises a (hetero)cycloalkane moiety, i.e., depicted as ––– The key of (hetero)cycloalkyl group is a single bond. (Hetero)cycloalkyl group may also be referred to as heterocycloalkyl group or cycloalkyl group, preferably cycloalkyl group, wherein the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornyl group, a (hetero)cycloheptyl group, a (hetero)cyclooctyl group, a (hetero)cyclononyl group or a (hetero)cyclodecyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropyl group, (hetero)cycloheptyl group or (hetero)cyclooctyl group, wherein the (hetero)cyclopropyl group, trans-(hetero)cycloheptyl group or (hetero)cyclooctyl group are optionally substituted. Preferably, Z comprises a cyclopropyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornene or norbornene group according to structure (Z46), a (hetero)cycloheptyl moiety according to structure (Z47), or a (hetero)cyclooctyl moiety according to structure (Z48). Here, Y 3 Selected from C(R 23 )2、NR 23 or O, where each R 23 Each is hydrogen, C1-C6 alkyl or optionally linked to L via a spacer, and is marked ––– The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropyl group is according to structure (Z49). In another preferred embodiment, the (hetero)cycloheptyl group is according to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group is according to structure (Z52), (Z53), (Z54), (Z55) or (Z56).

[0184]

[0185] Here, the R groups on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is typically selected from the structure (Zn)-(Zu), wherein the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring (Z44)-(Z56) fused to ring Z, and the carbon marked with * is directly connected to the peptide chain of the antibody. Preferably, ring Z is selected from (Zo)-(Zr). Since the linker Z in the context of this embodiment is formed by reaction with a (hetero)cycloalkene, it is depicted above as ––– The bond is a single bond.

[0186]

[0187] In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably by nucleophilic substitution or Michael addition, preferably by Michael addition. The preferred Michael reaction is a thiol-maleimide linkage, most preferably wherein Q is maleimide and F is a thiol group. Preferably, the thiol group is present in the side chain of a cysteine ​​residue. In a preferred embodiment, the linker Z comprises a succinimidyl ring or an open-ring succinamide derivative thereof. Preferred options for the linker Z include moieties selected from (Z57)-(Z71) depicted below.

[0188]

[0189] Herein, the wavy bonds marked with * are optionally connected to the antibody Ab via a linker, and the wavy bonds without a label are optionally connected to the payload via a linker. 29 It is C 1-12 Alkyl, preferably C 1-4 Alkyl, most preferably ethyl, and X 1 is O or S, preferably X 1 =0. The nitrogen atoms marked with ** in (Z67)-(Z71) correspond to the nitrogen atoms of the side chains of the antibody lysine residues. The carbon atoms of the phenyl groups in (Z69) and (Z70) are optionally substituted, preferably optionally fluorinated.

[0190] In a preferred embodiment, the linker group Z comprises a moiety selected from (Z1)-(Z71).

[0191] Connector L

[0192] The linker L connects the payload D to the linking group Z (in the conjugate according to structure (1)) or connects the payload D to the reactive group Q (in the compound according to structure (2)). Linkers are known in the art and may be cleavable or non-cleavable. The linker L preferably contains a self-immolative group or a cleavable linker comprising a peptide spacer and a p-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0193] In a preferred embodiment, the linker L is of the structure –(L 1 ) n –(L 2 ) o –(L 3 ) p –(L 4 ) q –, where (L 4 ) q Connected to payload D and (L 1 ) n Connect to Z or Q. In this article, L 1 , L 2 , L 3 and L 4 is a linker or connecting unit and each of n, o, p and q is independently 0 or 1, wherein n+o+p+q is at least 1. In a preferred embodiment, there is at least a linker L 1 and L 2 (i.e. n=1; o=1; p=0 or 1; q=0 or 1), more preferably there is a linker L 1 , L 2 and L 3 and the presence or absence of L 4 (i.e. n=1; o=1; p=1; q=0 or 1). In one embodiment, there is a linker L 1 , L 2 , L 3 and L 4 (ie n=1; o=1; p=1; q=1). In one embodiment, there is a linker L 1 , L 2 and L 3 And there is no L 4 (i.e. n=1; o=1; p=1; q=0).

[0194] Connectors, especially connectors L 1 , may contain one or more branch points for connecting multiple payloads to a single linker. In a preferred embodiment, the linker of the conjugate according to the present invention contains a branching portion. A "branching portion" in the context of the present invention refers to a portion embedded in the linker connecting three moieties. In other words, the branching portion contains 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. If a branching portion is present, it is preferably embedded in the linker L. 1 Among them, more preferably Sp 2 Part of or as NR 13Any moiety containing at least three bonds to other moieties is suitable as a branching moiety in the context of the present invention. In a preferred embodiment, the branching moiety is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring or a polycyclic moiety. Most preferably, the branching moiety is a nitrogen atom.

[0195] Connector L 1

[0196] Linker L is absent (n=0) or present (n=1) 1 Preferably, there is a linker L 1 And n = 1. 1 It can be selected from linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylidene group, C3-C 200 Cycloalkylene groups, 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 Optionally, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally, the groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, preferably selected from O, S(O) y’ and NR 21 The group consisting of, wherein y' is 0, 1 or 2, preferably y'=2, and R 21 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups.

[0197] In a preferred embodiment, the connector L 1 Contains polar groups. Such polar groups can be selected from (poly)ethylene glycol diamines (e.g. 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains and 1,z'-diaminoalkanes (wherein z' is the number of carbon atoms in the alkane, preferably z'=1-10), -(O)a –C(O)–NH–S(O)2–NR 13 – (as further defined below, see structure (23)), –C(S(O)3 (–) )–、–C(C(O)2 (–) )–、–S(O)2–、–P(O)2 (–) –, –O(CH2CH2O) t –, –NR 30 (CH2CH2NR 30 ) t – and the following two structures:

[0198]

[0199] The polar group may also contain an amino acid preferably selected from Arg, Glu, Asp, Ser and Thr. Here, for structure (23), a and R 13 Further defined as follows. t is an integer in the range of 0 to 15, preferably 1 to 10, more preferably 2 to 5, most preferably t=2 or 4. Each R 30 Independently H, C 1-12 Alkyl, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl. Linker L 1 There may be more than one such polar group, for example at least two polar groups. Polar groups may also be present in the linker L 1 In the branches of the present invention, which branch from the branching moiety as defined elsewhere. Preferably, nitrogen or carbon atoms are used as branching moieties. Particularly preferably, -O(CH2CH2O) is present in the branches. t –Polar groups.

[0200] In a preferred embodiment, the connector L 1 is or comprises a sulfonamide group, preferably a sulfonamide group according to structure (23):

[0201]

[0202] The wavy line represents the connection to the rest of the compound, usually to Q and L. 2 , L 3 , L 4 or D, preferably with Q and L 2 Preferably, (O) a The C(O) moiety is connected to Q and NR 13 Partially connected to L 2 , L 3 , L 4 or D, preferably connected to L2 .

[0203] In structure (23), a=0 or 1, preferably a=1, and R 13 Selected from hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The group consisting of (hetero)arylalkyl groups, the C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally replaced by one or more selected from O, S and NR 14 The heteroatom interval, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is via a spacer moiety (preferably Sp as defined below) 2 ) is connected to N, in one embodiment, D is connected via – (B) e –(A) f –(B) g –C(O)-connected to N.

[0204] In a preferred embodiment, R 13 is hydrogen or C1-C 20 Alkyl groups, more preferably R 13 is hydrogen or C1-C 16 Alkyl groups, even more preferably R 13 is hydrogen or C1-C 10 Alkyl groups, wherein the alkyl group is optionally substituted and optionally substituted with one or more selected from O, S and NR 14 The heteroatom, preferably O, is interrupted, wherein R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 13 In another preferred embodiment, R 13 It is C1-C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 alkyl group, wherein the alkyl group is optionally interrupted by one or more O atoms, and wherein the alkyl group is optionally substituted by an -OH group, preferably a terminal -OH group. In this embodiment, it is further preferred that R13 is a (poly)ethylene glycol chain containing a terminal -OH group. In another preferred embodiment, R 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and isopropyl, and even more preferably selected from the group consisting of hydrogen, methyl and ethyl. Still even more preferably, R 13 is hydrogen or methyl, and most preferably R 13 It's hydrogen.

[0205] In a preferred embodiment, L 1 According to the structure (24):

[0206]

[0207] In this paper, a and R 13 As defined above, Sp 1 and Sp 2 is independently a spacer moiety and b and c are independently 0 or 1. Preferably, b = 0 or 1 and c = 1, more preferably b = 0 and c = 1. In one embodiment, the spacer Sp 1 and Sp 2 Independently selected from linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylidene group, C3-C 200 Cycloalkylene groups, 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 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 is optionally substituted and optionally substituted by one or more radicals selected from O, S and NR 16 The heteroatom interval, where R 16 independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24The group consisting of an alkyl radical, an alkenyl radical, an alkynyl radical and a cycloalkyl radical is optionally substituted. When an alkylene radical, an alkenylene radical, an alkynylene radical, a cycloalkyl radical, a cycloalkenylene radical, a cycloalkynylene radical, an alkylarylene radical, an arylalkylene radical, an arylalkenylene radical and an arylalkynylene radical are interrupted by one or more heteroatoms as defined above, preferably said radicals are interrupted by one or more O atoms and / or by one or more SS groups.

[0208] More preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylidene group, C3-C 100 Cycloalkylene groups, 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 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 is optionally substituted and optionally substituted by one or more radicals selected from O, S and NR 16 The heteroatom interval, where R 16 independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0209] Even more preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylidene group, C3-C 50 Cycloalkylene groups, C5-C 50 Cycloalkenylene group, C8-C 50 Cycloalkynylene group, C7-C 50Alkyl arylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene groups and C9-C 50 The group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group and an arylalkynylene group is optionally substituted and optionally substituted by one or more radicals selected from O, S and NR 16 The heteroatom interval, where R 16 independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0210] Still even more preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylidene group, C3-C 20 Cycloalkylene groups, 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 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 is optionally substituted and optionally substituted by one or more radicals selected from O, S and NR 16 The heteroatom interval, where R 16 independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0211] In these preferred embodiments, it is further preferred that the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group are unsubstituted and optionally substituted by one or more groups selected from O, S and NR. 16 The group consisting of heteroatoms, preferably O, wherein R 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0212] Most preferably, the spacer moiety Sp 1 and Sp 2 , if present, are independently selected from linear or branched C1-C 20 The group consisting of an alkylene group, the alkylene group is optionally substituted and optionally substituted by one or more selected from the group consisting of O, S and NR 16 The heteroatom-interval consisting of a group wherein R 16 independently selected from hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The group consisting of alkyl, alkenyl, alkynyl and cycloalkyl groups, wherein the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. In this embodiment, it is further preferred that the alkylene group is unsubstituted and optionally substituted with one or more selected from O, S and NR 16 The heteroatoms (preferably O and / or SS) are separated, wherein R 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0213] Preferred spacer moieties Sp 1 and Sp 2 Therefore, including -(CH2) r -、-(CH2CH2) r -、-(CH2CH2O) r -、-(OCH2CH2) r -、-(CH2CH2O) r CH2CH2-, -CH2CH2(OCH2CH2) r -、-(CH2CH2CH2O) r -、-(OCH2CH2CH2) r -、-(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r-, wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20 and still 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.

[0214] Alternatively, the preferred linker L 1 Can be controlled by –(W) k –(A) d –(B) e –(A) f –(C(O)) g – means, where:

[0215] - d = 0 or 1, preferably d = 1;

[0216] - e = an integer in the range of 0 to 10, preferably e = 0, 1, 2, 3, 4, 5 or 6, preferably an integer in the range of 1 to 10, most preferably e = 1, 2, 3 or 4;

[0217] - f = 0 or 1, preferably f = 0;

[0218] - wherein d+e+f is at least 1, preferably in the range of 1 to 5; and preferably, wherein d+f is at least 1, preferably d+f=1.

[0219] - g = 0 or 1, preferably g = 1;

[0220] - k = 0 or 1, preferably k = 1;

[0221] -A is a sulfonamide group according to structure (23);

[0222] -B is a –CH2–CH2–O– or –O–CH2–CH2– moiety, or (B) e is –(CH2–CH2–O) e1 –CH2–CH2–

[0223] or –(CH2–CH2–O) e1 –CH2– moiety, where e1 is defined the same as e;

[0224] -W is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–,

[0225] –C(O)(CH2) m C(O)–, –C(O)(CH2)m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably wherein W is -OC(O)NH-, -C(O)(CH2) m C(O)NH— or —C(O)NH—, and wherein m is an integer in the range of 0 to 10, preferably m=0, 1, 2, 3, 4, 5 or 6, most preferably m=2 or 3;

[0226] - preferably, wherein L 1 Via(W) k Connected to Q and via (C(O)) g , preferably connected to L via C(O) 2 , L 3 , L 4 or D, preferably connected to L 2 .

[0227] In the context of this example, the wavy line in structure (23) indicates that the adjacent groups, such as (W) k (B) e and (C(O)) g Preferably A is according to structure (23), wherein a=1 and R 13 =H or C1-C 20 Alkyl groups, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0228] Preferred linker L 1 With structure – (W) k –(A) d –(B) e –(A) f –(C(O)) g -,in:

[0229] (a) k=0; d=1; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2.

[0230] (b) k = 1; W = –C(O)(CH2) m C(O)NH–;m=2;d=0;(B) e =

[0231] –(CH2–CH2–O) e1 –CH2–CH2–; f=0; g=1; e1=1, 2, 3 or 4, preferably e=1.

[0232] (c)k=1; W=–OC(O)NH–; d=0; B=–CH2–CH2–O–; g=1; f=0; e=1, 2,

[0233] 3 or 4, preferably e=2.

[0234] (d) k = 1; W = –C(O)(CH2) m C(O)NH–;m=2;d=0;(B) e =

[0235] –(CH2–CH2–O) e1 –CH2–CH2–; f=0; g=1; e1=1, 2, 3 or 4, preferably e1=4.

[0236] (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.

[0237] (f)k=1; W=–(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, m=3; d=0; (B) e =

[0238] –(CH2–CH2–O) e1 –CH2–CH2–; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4.

[0239] (g) k=0; d=0; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2.

[0240] (h) k=1; W=—C(O)NH—; d=0; g=1; f=0; B=—CH 2 —CH 2 —O—; e=1, 2, 3 or 4, preferably e=2.

[0241] In a preferred embodiment, the connector L 1 Contains branched nitrogen atoms located between Q or Z and (L 2 ) o and contains an additional moiety D as a substituent, which is preferably connected to the branching nitrogen atom via a linker. An example of a branching nitrogen atom is the nitrogen atom NR in structure (23) 13 , where R 13 is connected to the second occurrence of D via a spacer moiety. Alternatively, the branched nitrogen atom may be located according to the structure -(W) k –(A)d –(B) e –(A) f –(C(O)) g –L 1 In one embodiment, L 1 By – (W) k –(A) d –(B) e –(A) f –(C(O)) g –N*[–(A) d –(B) e –(A) f –(C(O)) g’ –]2 represents, wherein A, B, W, d, e, f, g and k are as defined above and are selected individually at each occurrence, and N* is a branching nitrogen atom, –(A) d –(B) e –(A) f –(C(O)) g’ Both instances of – are connected to it. Here, both (C(O)) g’ Parts are connected to –(L 2 ) o –(L 3 ) p –(L 4 ) q –D, where L 2 , L 3 , L 4 , o, p, q and D are as defined above and are each selected individually. In a preferred embodiment, L 2 , L 3 , L 4 , o, p, q and D for each of (C(O)) g’ The two parts of the connection are the same.

[0242] Preferred linkers L containing branched nitrogen atoms 1 With structure – (W) k –(A) d –(B) e –(A) f –(C(O)) g –N*[–(A') d’ –(B') e’ –(A') f’ –(C(O)) g ”–]2, where:

[0243] (i) k=d=g=e'=1;f=d'=g'=0;W=–C(O)–;B=B'=–CH2–CH2–O–;A is according to structure (23), where a=0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0244] (j) k = d = g = e' = g' = 1; f = d' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is according to structure (23), where a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0245] Connector L 2

[0246] The linker L is absent (o=0) or present (o=1) 2 Preferably, there is a linker L 2 And o=1. Joint L 2 is a peptide spacer. The peptide spacer is preferably composed of (NH-CR 17 –CO) n Definition, where R 17Represents an amino acid side chain known in the art. Here, the amino acid can be a natural or synthetic amino acid. Preferably, the amino acids are all in the L-configuration. n is an integer in the range of 1 to 5, preferably an integer in the range of 2 to 5. Therefore, the peptide spacer preferably contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), a tripeptide (n=3) or a tetrapeptide (n=4), and most preferably the peptide spacer is a dipeptide. Although any peptide spacer can be used, it is preferred that the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-L In one embodiment, AcLys is acetyl-lysine, and iGlu is isoglutamic acid. In one embodiment, L 2 =Val-Cit. In one embodiment, L 2 =Val-Ala.

[0247] R 17 R represents an amino acid side chain, preferably selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, 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 those of Val, Cit, Ala, Glu and Lys. Or in other words, R 17Preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred examples are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu) and CH(CH3)2(Val). Most preferably, R 17 It is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys) or CH(CH3)2(Val).

[0248] In a particularly preferred embodiment, the peptide spacer can be represented by the general structure (L3):

[0249]

[0250]

[0251] In this paper, R 17 As defined above, preferably R 17 Is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates the same as (L 1 ) n and (L 3 ) p connection, preferably according to L of structure (L3) 2 Through NH and (L 1 ) n Connected and connected via C(O) and (L 3 ) p connect.

[0252] Connector L 3

[0253] The linker L is absent (p=0) or present (p=1) 3 Preferably, there is a linker L 3 And p = 1. Connector L 3is a self-cleavable spacer, also known as a self-eliminating spacer. 3 is a p-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L4):

[0254]

[0255] In this article, the wavy line indicates the connection with Q or Z, L 1 or L 2 And with L 4 Or D. Usually, PABC derivatives are connected via NH to Q, Z, L 1 or L 2 connection, preferably with L 2 Connected, and through O and L 4 or D connection.

[0256] A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole, and furan. Suitable 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,4-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0257] R 21 Selected from H, R 26 , C(O)OH and C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl groups, C3–C 10 Alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally replaced by one or more selected from O, S and NR 28 The heteroatom interval, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 It is C3–C 10 (Hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or –(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably -(CH2CH2O) s H, wherein s is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s=1, 2, 3 or 4. More preferably, R 21 is H or C(O)R26 , where R 26 =4-methyl-piperazine or morpholine. Most preferably, R 21 It’s H.

[0258] Connector L 4

[0259] The linker L is absent (q=0) or present (q=1) 4 Preferably, there is a linker L 4 And q=1. Connector L 4 Selected from:

[0260] - According to the structure –NR 22 –(C z -alkylene)-C(O)-aminoalkanoic acid spacer, wherein z is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl;

[0261] - According to the structure –NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -C(O)-, wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R 22 is H or C1–C4 alkyl; and

[0262] -According to the structure –NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer, wherein h is 0 or 1, z is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl.

[0263] Connector L 4 It can be an aminoalkanoic acid spacer, i.e. –NR 22 –(C z -alkylene)-C(O)-, wherein z is an integer in the range of 1 to 20, preferably 1 to 10, most preferably 1 to 6. Herein, the aminoalkanoic acid spacer is typically attached to L via a nitrogen atom. 3 and connected to D via the carbonyl moiety. Preferred linkers L 4 is selected from 6-aminohexanoic acid (Ahx, z=5), β-alanine (z=2) and glycine (Gly, z=1), even more preferably 6-aminohexanoic acid or glycine. In one embodiment, L 4 =6-aminohexanoic acid. In one embodiment, L 4 = glycine. Here, R 22is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 It’s H.

[0264] Alternatively, connector L 4 It can be based on the structure –NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -(C(O)-)ethylene glycol spacer, wherein e6 is an integer in the range of 1 to 10, preferably e6 is in the range of 2 to 6, and e7 is an integer in the range of 1 to 3, preferably e7 is 2. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 It’s H.

[0265] Alternatively, connector L 4 It can be based on the structure –NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer wherein h is 0 or 1, z is an integer in the range of 1 to 20, preferably an integer in the range of 2 to 6, even more preferably z=2 or 5, most preferably z=2. 22 is H or C1–C4 alkyl. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 Herein, h is preferably 1, in which case the linker L 4 It is particularly suitable for conjugation via the phenolic hydroxyl groups present on the payload D.

[0266] Payload D

[0267] D represents a target molecule D that is or will be linked to the antibody, which is also referred to in the art as a payload. D is selected from anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines. Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs. Alternatively, D is defined as a pharmaceutically active substance, such as an anticancer agent, preferably a cytotoxin. Preferably, D is selected from anthracyclines, camptothecins, maytansinoids, enediynes, amanitins, auristatins and pyrrolobenzodiazepines. In one embodiment, D is an enediyne. In one embodiment, D is an auristatin. In one embodiment, D is a camptothecin. Most preferably, D is a camptothecin.

[0268] In a preferred embodiment, the enediyne is selected from the group consisting of calicheamicin, esperamicin, shishijimicin and namenamicin, more preferably calicheamicin. In another preferred embodiment, the auristatin is selected from the group consisting of MMAD, MMAE, MMAF or PF-06380101, more preferably MMAE or PF-06380101. In another preferred embodiment, the camptothecin is selected from the group consisting of Figure 6 The compound shown is preferably selected from the group consisting of topotecan, silatecan, cositecan, exotecan, exotecan-S, DXd, SN-38, lertotecan, gimatecan, belotecan, rubitecan, AMDCPT and G-AMDCPT, more preferably exotecan or DXd, most preferably exotecan.

[0269] In a particularly preferred embodiment, D is selected from the group consisting of calicheamicin, MMAE, PF-06380101, exitecan and DXd, more preferably selected from the group consisting of calicheamicin, MMAE and exitecan, and most preferably, D is exitecan.

[0270] Compounds according to general structure (2) may contain more than one moiety D. When more than one cytotoxin D is present, the cytotoxin D may be the same or different, typically they are the same. In a preferred embodiment, the compound according to general structure (2) contains 1 or 2 occurrences of D, most preferably 2 occurrences of D. Typically, the second occurrence of D is present in a linker L, which may contain a branching moiety, typically a branching nitrogen atom, which is connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branching moiety via the same linker. Similarly, antibody conjugates according to structure (1) may contain more than one moiety D per linker Z.

[0271] Preferred antibody conjugates

[0272] Preferred antibody conjugates according to the first aspect are selected from the group consisting of compounds (I)-(III), more preferably (II) or (III), most preferably (II). More preferred antibody conjugates are selected from (X)-(XIII). In a particularly preferred embodiment, the antibody conjugate is selected from (Xa), (XIb), (XIIg), (XIIh) and (XIIIe). In an even more preferred embodiment, the antibody conjugate is selected from (XI) and (XIII), more preferably (XIb) or (XIIIe), more preferably the antibody conjugate is according to (XIII), most preferably according to (XIIIe). The structures of these antibody conjugates are defined below.

[0273] Antibody conjugate (I) has the following structure:

[0274] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–(L 4 ) q –D} x ] y

[0275] (I)

[0276] in:

[0277] -AB, L 6 , Z, D, x and y are as defined above;

[0278] -L 1 Is caused by – (A) d –(B) e –(A) f –(C(O)) g - a linker denoted as defined above;

[0279] -L 2 It is Val-Cit or Val-Ala;

[0280] -L 3 It is a PABC derivative based on structure (L4);

[0281] -L 4 Yes–N–(C z -alkylene)–C(O)– or –NR 22 –(C z -alkylene)–NR 22 –, where z and R 22 As defined above;

[0282] -q=0 or 1.

[0283] In the case of antibody conjugate (I), preferably for L 1 , d = 1 (according to A of structure (23), preferably a = 1 and R 13 =H), e=2, f=0 and g=1. In the case of antibody conjugate (I), preferably L 2 =Val-Cit. In the case of antibody conjugate (I), preferably, for L 3 , R 21 =H. In the case of the antibody conjugate (I), preferably, if q=1, then z=1 or 5.

[0284] In a particularly preferred embodiment, the antibody conjugate according to structure (I) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0285] Antibody conjugate (II) has the following structure:

[0286] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–D} x ] y

[0287] (II)

[0288] in:

[0289] -AB, L 6 , Z, D, x and y are as defined above;

[0290] -L 1 It is composed of –(A)–(B) e –(C(O))– represented by a linker, as defined above;

[0291] -L 2 It is Val-Cit or Val-Ala;

[0292] -L 3 is a PABC derivative according to structure (L4), wherein R 21 =H.

[0293] In the case of antibody conjugate (II), preferably, for L 1 , e=2, according to structure (23), preferably, a=1 and R 13 =H. In the case of antibody conjugate (II), preferably L 2=Val-Cit.

[0294] In a particularly preferred embodiment, the antibody conjugate according to structure (II) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0295] Antibody conjugate (III) has the following structure:

[0296] AB–[(L 6 )–{Z–(L 1 )–(L 2 )–(L 3 )–(L 4 )–D} x ] y

[0297] (III)

[0298] in:

[0299] -AB, L 6 , Z, D, x and y are as defined above;

[0300] -L 1 It is composed of –(A)–(B) e –(C(O))– represented by a linker, as defined above;

[0301] -L 2 It is Val-Cit or Val-Ala;

[0302] -L 3 is a PABC derivative according to structure (L4), wherein R 21 =H;

[0303] -L 4 Yes – NR 22 –(C z -alkylene)–NR 22 –, where R 22 As defined above and z is an integer in the range of 1 to 6.

[0304] In the case of antibody conjugate (III), preferably, for L 1 , e=2, and a=1 and R 13 =H. In the case of antibody conjugate (III), preferably, L 2 =Val-Cit. In the case of the antibody conjugate (III), preferably z=2.

[0305] In a particularly preferred embodiment, the antibody conjugate according to structure (III) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0306] The antibody conjugate (X) has a linker payload portion according to the following structure:

[0307]

[0308] in:

[0309] - The wavy line indicates the connection to Z;

[0310] -L 2 , o and D are as defined above.

[0311] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (Xa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (Xb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (X) preferably has structure (Xa).

[0312] In a particularly preferred embodiment, the antibody conjugate according to structure (X) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0313] The antibody conjugate (XI) has a linker payload portion according to the following structure:

[0314]

[0315] in:

[0316] - The wavy line indicates the connection to Z;

[0317] -L 2 , o and D are as defined above.

[0318] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (XIa), L2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (XI) preferably has structure (XIb).

[0319] In a particularly preferred embodiment, the antibody conjugate according to structure (XI) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably calicheamicin as the payload D.

[0320] The antibody conjugate (XII) has a linker payload portion according to the following structure:

[0321]

[0322] in:

[0323] - The wavy line indicates the connection to Z;

[0324] -L 2 , L 4 , o, q and D are as defined above.

[0325] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (XIIa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2.

[0326] L may or may not be present 4 For the preferred antibody conjugate (XIIc), q=0 and L 4 For the preferred antibody conjugate (XIId), q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0327] For the preferred antibody conjugate (XIIe), L 2 According to the structure (L3), R 17 is CH3, q=0 and L 4 For the preferred antibody conjugate (XIIf), L 2 According to the structure (L3), R 17 is CH3, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 10, preferably wherein z=2, and R 22 is H or C1–C4 alkyl.

[0328] For the preferred antibody conjugate (XIIg), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0 and L 4 For the preferred antibody conjugate (XIIh), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0329] In the case of the antibody conjugate (XII), preferably z is an integer in the range of 1 to 10, more preferably z = 2 to 6, most preferably z = 2. In the case of the antibody conjugate (XII), further preferably R 22 It is H or CH3.

[0330] In the case of antibody conjugate (XII), structures (XIIg) and (XIIh) are most preferred.

[0331] In a particularly preferred embodiment, the antibody conjugate according to structure (XII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0332] The antibody conjugate (XIII) has a linker payload portion according to the following structure:

[0333]

[0334] in:

[0335] - The wavy line indicates the connection to Z;

[0336] -L 2 , L 4 , o, q and D are as defined above.

[0337] L may or may not be present 2 , preferably there is L 2 and o = 1. For the preferred antibody conjugate (XIIIa), L 2 According to the structure (L3) and R 17 is CH3. For the preferred antibody conjugate (XIIIb), L 2 According to the structure (L3) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH3.

[0338] L may or may not be present 4 For the preferred antibody conjugate (XIIIc), q=0 and L 4 For the preferred antibody conjugate (XIIId), q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0339] For the preferred antibody conjugate (XIIIe), L 2 According to the structure (L3), R 17 is CH3, q=0 and L 4 For the preferred antibody conjugate (XIIIf), L 2 According to the structure (L3), R 17 is CH3, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 10, preferably wherein z=2, and R 22 is H or C1–C4 alkyl.

[0340] For the preferred antibody conjugate (XIIIg), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0 and L 4 For the preferred antibody conjugate (XIIIh), L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1 and L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 - a diamine spacer, wherein z is an integer in the range of 1 to 20, and R 22 is H or C1–C4 alkyl.

[0341] In the case of the antibody conjugate (XIII), preferably z is an integer in the range of 1 to 10, more preferably z = 2 to 6, most preferably z = 2. In the case of the antibody conjugate (XIII), further preferably R 22 It is H or CH3.

[0342] In the case of antibody conjugate (XIII), structure (XIIIe) is most preferred.

[0343] In a particularly preferred embodiment, the antibody conjugate according to structure (XIII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exotecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE and exotecan, and most preferably exotecan as the payload D.

[0344] In a particularly preferred embodiment, the antibody conjugate according to the invention is according to structure (XIb) as defined above, and the antibody is cofiluzumab as defined above, and the payload is calicheamicin.

[0345] In a particularly preferred embodiment, the antibody conjugate according to the invention is according to structure (XIIIe) as defined above, and the antibody is cofetizumab as defined above, and the payload is exetitecan.

[0346] It is further preferred that these preferred antibody conjugates are conjugated via a glycan, i.e. b = 1, more preferably a trimmed glycan, i.e. j = 0. Here, it is further preferred that S = GalNAc and w' = 0. Here, it is further preferred that the linker Z is formed by an azide-alkyne cycloaddition, preferably the linker Z = (Z39), wherein ring Z = (Za) and V = CH2. Here, it is further preferred that x = 1. Here, it is further preferred that y = 2, more preferably x = 1 and y = 2.

[0347] In the most preferred embodiment, the antibody conjugate according to the present invention is according to structure (XIIIe) as defined above, and the antibody is ennoizumab YTE as defined above and the payload is ixetine, wherein b=1, e=0, S=GalNAc, w'=0, linker Z=(Z39), wherein ring Z=(Za) and V=CH2, x=1 and y=2.

[0348] Compounds according to formula (2)

[0349] The compound has the general structure (2):

[0350] Q–L–D (2)

[0352] in:

[0353] -Q is the reactive moiety;

[0354] -L is the linker connecting Z to D;

[0355] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0356] Compounds of general structure (2) may also be referred to as "linker-drug constructs" for the linker L and payload D of the final conjugate. Compounds according to general formula (2) can be prepared by one skilled in the art using standard organic synthesis techniques, and as illustrated in the Examples. Linker L and payload D are defined in the context of the conjugate of structure (1).

[0357] Reactive part Q

[0358] Compounds according to general structure (2) contain a reactive moiety Q. In the context of the present invention, the term "reactive moiety" may refer to a chemical moiety comprising a reactive group, but may also refer to the reactive group itself. For example, a cyclooctynyl group is a reactive group comprising a reactive group, i.e., a C-C triple bond. Similarly, an N-maleimido group is a reactive group comprising a C-C double bond as a reactive group. However, reactive groups, such as an azide reactive group, a sulfhydryl reactive group, or an alkynyl reactive group, may also be referred to herein as a reactive moiety.

[0359] Q is used to connect to S(F) x In other words, Q is reactive toward and complementary to F. Herein, a reactive group is said to be "complementary" to a reactive group when it selectively reacts with the reactive group, optionally in the presence of other functional groups. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Thus, compounds according to general structure (2) are conveniently used in conjugation reactions, wherein a chemical reaction occurs between Q and F to form an antibody conjugate comprising a covalent linkage between the payload D and the antibody.

[0360] The exact nature of Q and F depends on the type of conjugation reaction adopted. Those skilled in the art will be able to select an appropriate combination of Q and F. Preferably, Q and F are reactive in cycloadditions or nucleophilic reactions. Therefore, Q preferably comprises a click probe, a sulfhydryl, a sulfhydryl reactive moiety, an amine or an amine reactive moiety, more preferably Q is a click probe, a sulfhydryl reactive moiety or an amine reactive moiety, most preferably Q is a click probe. The click probe is reactive in cycloadditions (click reactions) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxythiophenes, sydney ketones, olefin moieties and alkyne moieties. Preferably, the click probe comprises or is an olefin moiety or an alkyne moiety, more preferably wherein the olefin is a (hetero)cycloolefin and / or the alkyne is a terminal alkyne or a (hetero)cycloalkyne. Typical sulfhydryl reactive moieties are selected from maleimide moiety, haloacetamide moiety, propadiene amide moiety, phosphinamide moiety, cyanoacetyl moiety, vinyl sulfone, vinyl pyridine moiety or methylsulfonylphenyl oxadiazole moiety. Most preferably, the sulfhydryl reactive moiety comprises or is a maleimide moiety. Typical amine reactive moieties are selected from N-hydroxysuccinimide ester and other activated esters, p-nitrophenyl carbonate and other activated carbonates, isocyanates, isothiocyanates, haloacetamides and benzyl halides. In a preferred embodiment, Q is selected from olefin moiety, alkyne moiety, sulfhydryl reactive moiety or amine reactive moiety, more preferably olefin moiety or alkyne moiety, even more preferably alkyne moiety. Herein, olefin is preferably (hetero) cycloalkene, and alkyne is preferably terminal alkyne or (hetero) cycloalkyne. Most preferably, Q is cyclic (hetero) alkyne moiety. Each of these parts is further defined below.

[0361] Therefore, in a particularly preferred embodiment, Q comprises cyclic (hetero) alkyne moiety.Alkynyl group can also be referred to as (hetero) cycloalkynyl group, i.e. heterocycloalkynyl group or cycloalkynyl group, wherein (hetero) cycloalkynyl group is optionally substituted.Preferably, (hetero) cycloalkynyl group is (hetero) cycloheptynyl group, (hetero) cyclooctynyl group, (hetero) cyclononyl group or (hetero) cyclodecynyl group.Herein, (hetero) cycloalkyne can be optionally substituted.Preferably, (hetero) cycloalkynyl group is optionally substituted (hetero) cycloheptynyl group or optionally substituted (hetero) cyclooctynyl group.Most preferably, (hetero) cycloalkynyl group is (hetero) cyclooctynyl group, wherein (hetero) cyclooctynyl group is optionally substituted.

[0362] In a particularly preferred embodiment, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and is according to structure (Q1):

[0363]

[0364] In this article:

[0365] -Depicted as– – – The bond is a double bond or a triple bond;

[0366] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0367] –C 24 a group consisting of (hetero)arylalkyl groups;

[0368] -Y 2 It 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 (-) Balanced cationic sulfur atoms, where B (-) is an anion, and each R 31 R alone 15 or a connection to D, which is connected via L;

[0369] -u is 0, 1, 2, 3, 4, or 5;

[0370] - u' is 0, 1, 2, 3, 4 or 5, wherein u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0371] -v=an integer in the range of 0 to 16;

[0372] Usually, v = (u + u') x 2 (when passing through Y 2when connected to L (represented by a wavy bond)) or [(u+u')x 2]-1 (when connected to L via one of the carbon atoms (represented by a wavy bond)).

[0373] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1a):

[0374]

[0375]

[0376] In this article,

[0377] -R 15 and Y 2 As defined above

[0378] -u is 0, 1, 2, 3, 4, or 5;

[0379] - u' is 0, 1, 2, 3, 4 or 5, where u + u' = 4, 5, 6, 7 or 8;

[0380] -v=an integer in the range of 8 to 16.

[0381] In a preferred embodiment, u+u'=4, 5 or 6, more preferably u+u'=5. In a preferred embodiment, v=8, 9 or 10, more preferably v=9 or 10, most preferably v=10.

[0382] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2)-(Q20) and (Q20a) depicted below.

[0383]

[0384] Herein, the connection to L depicted by the wavy bond can be to any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) can have a connection to L, or can contain a hydrogen atom or be optionally functionalized. (–) is an anion, which is preferably selected from (–) OTf、Cl (–) Br (–) or I (–) , most preferably B (–) yes (–) OTf. In the conjugation reaction, B (–) does not need to be a pharmaceutically acceptable anion, since B (–)In any case, exchange will take place with anions present in the reaction mixture. In case (Q19) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable when isolating the conjugate according to the invention, making the conjugate readily useful as a drug.

[0385] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21)-(Q38) and (Q38a) depicted below.

[0386]

[0387] In structure (Q38), B (–) is an anion, which is preferably selected from (–) OTf、Cl (–) Br (–) or I (–) , most preferably B (–) yes (–) OTf.

[0388] In a preferred embodiment, Q comprises a (hetero)cyclooctyne moiety or a (hetero)cycloheptyne moiety, preferably according to Structure (Q8), (Q26), (Q27), (Q28), (Q37) or (Q38a), more preferably according to Structure (Q8), (Q26), (Q27), (Q28) or (Q37), which is optionally substituted. Each of these preferred options for Q is further defined below.

[0389] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is unsubstituted.

[0390] In alternative preferred embodiments, Q comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also known as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q39) shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q is structure (Q42) as further defined below.

[0391] In alternative preferred embodiments, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also known as a DIBO, DIBAC, DBCO or ADIBO group, which is optionally substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 independently selected from hydrogen, linear or branched C1-C 12 Alkyl group or C4-C 12 The aromatic ring in (Q40) is optionally O-sulfonylated at one or more positions, while the ring 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 structure (Q43) as further defined below.

[0392] In an alternative preferred embodiment, Q comprises a heterocycloheptenyl group and is according to structure (Q37).

[0393]

[0394] In a particularly preferred embodiment, Q comprises a cyclooctynyl group and is according to structure (Q42):

[0395]

[0396] In this article:

[0397] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24Alkyl (hetero)aryl groups and C7–

[0398] C 24 a group consisting of (hetero)arylalkyl groups;

[0399] -R 18 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 a group consisting of (hetero)arylalkyl groups;

[0400] -R 19 Selected from hydrogen, halogen, C1–C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, said alkyl groups being optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Q or D linked via a spacer moiety; and

[0401] -l is an integer in the range 0 to 10.

[0402] In a preferred embodiment of the reactive group according to Structure (Q42), R 15 are independently selected from hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 Independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably two R 18 are all H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 is H. In a preferred embodiment of the reactive group according to Structure (Q42), l is 0 or 1, more preferably l is 1.

[0403] In a particularly preferred embodiment, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43):

[0404]

[0405] In this article:

[0406] -R 15 are independently selected from hydrogen, halogen, -OR 16 、-NO2、-CN、-S(O)2R 16 、-S(O)3 (-) , C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero)aryl groups and C7–C 24 The group consisting of (hetero)arylalkyl groups, and wherein the alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, C6–C 24 (Hetero)aryl groups, C7–C 24 Alkyl (hetero) aryl groups and C7

[0407] –C 24 a group consisting of (hetero)arylalkyl groups;

[0408] -Y is N or CR 15 .

[0409] In a preferred embodiment of the reactive group according to Structure (Q43), R 15 are independently selected from hydrogen, halogen, -OR 16 、-S(O)3 (-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from hydrogen and -S(O)3 (-) In a preferred embodiment of the reactive group according to Structure (Q43), Y is N or CH, more preferably Y═N.

[0410] In particularly preferred embodiments, Q comprises a heterocycloheptenyl group and is according to structure (Q37) or (Q38a), preferably according to structure (Q37)

[0411]

[0412] In an alternative preferred embodiment, Q comprises cycloalkene moiety.Alkenyl Q also can be referred to as (hetero) cycloalkenyl group, i.e. heterocycloalkenyl group or cycloalkenyl group, preferably cycloalkenyl group, wherein this (hetero) cycloalkenyl group is optionally substituted.Preferably, (hetero) cycloalkenyl group is (hetero) cyclopropenyl group, (hetero) cyclobutenyl group, norbornenyl group, norbornadienyl group, trans-(hetero) cycloheptenyl group, trans-(hetero) cyclooctenyl group, trans-(hetero) cyclononenyl group or trans-(hetero) cyclodecenyl group, and they all can be optionally substituted.Particularly preferred is (hetero) cyclopropenyl group, trans-(hetero) cycloheptenyl group or trans-(hetero) cyclooctenyl group, wherein this (hetero) cyclopropenyl group, trans-(hetero) cycloheptenyl group or trans-(hetero) cyclooctenyl group is optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). Here, Y 3 Selected from C(R 23 )2、NR 23 or O, where each R 23 Each is hydrogen, C1-C6 alkyl or optionally linked to L via a spacer, and is marked ––– The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropenyl group is according to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctenene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).

[0413]

[0414]

[0415] Here, the R group on Si in (Q50) and (Q51) is generally an alkyl group or an aryl group, preferably a C1-C6 alkyl group.

[0416] In another preferred embodiment, Q is a sulfhydryl reactive probe. In this embodiment, Q is a reactive group compatible with cysteine ​​conjugation. Such probes are known in the art and can be selected from the group consisting of a maleimide moiety, a haloacetamide moiety, a propadienamide moiety, a phosphinamide moiety, a cyanoethynyl moiety, a vinyl sulfone, a vinyl pyridine moiety, or a methylsulfonylphenyl oxadiazole moiety. Most preferably, Q comprises or is a maleimide moiety. The reagent can be a monoalkylated type or can be a cross-linking agent for reacting with two cysteine ​​side chains.

[0417] In a further preferred embodiment, probe Q is selected from the group consisting of (Q57)-(Q71) depicted below.

[0418]

[0419] in:

[0420] -X 6 is H, halogen, PhS, MeS, preferably halogen, such as Cl, Br, I;

[0421] -X 7 is halogen, PhS, MeS, preferably halogen, such as Cl, Br, I;

[0422] -R 24 Is H or C 1-12 Alkyl, preferably H or C 1-6 alkyl;

[0423] -R 25 It is H, C 1-12 Alkyl group, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl, preferably H or p-methylphenyl;

[0424] - wherein the aromatic rings of (Q61) and (Q63) may optionally be heteroaromatic rings, such as phenyl or pyridine rings.

[0425] In a preferred embodiment of the thiol-reactive probe (Q57), the probe Q is selected from the group consisting of (Q72)-(Q74) depicted below.

[0426]

[0427] in:

[0428] -R 27 It is C 1-12 Alkyl group, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl;

[0429] -t is an integer in the range of 0 to 15, preferably 1 to 10.

[0430] In another preferred embodiment, Q is an amine reactive probe. In this embodiment, Q is a reactive group compatible with lysine conjugation. Such probes are known in the art and can be selected from the group consisting of N-hydroxysuccinimide groups, isocyanate groups, isothiocyanate groups, and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimide ester or p-nitrophenyl carbonate moiety.

[0431] In a further preferred embodiment, probe Q is selected from the group consisting of (Q75)-(Q79) depicted below.

[0432]

[0433] Among them, X 2 is halogen, preferably F.

[0434] In a preferred embodiment, Q is selected from the group consisting of (Q1)-(Q79).

[0435] Antibodies according to the general structure (3)

[0436] The antibody has the general structure (3):

[0437] AB–[(L 6 ) b –{F} x ] y (3)

[0439] in:

[0440] -AB is an antibody that can target tumors expressing PTK7;

[0441] -b is 0 or 1;

[0442] -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–;

[0443] -F is the reactive moiety;

[0444] -x is 1 or 2; and

[0445] -y is 1, 2, 3, or 4.

[0446] The antibody of general structure (3) may also be referred to as a "(modified) antibody" because it is an antibody containing a reactive group F, wherein the reactive group F is naturally occurring or the antibody is modified to incorporate the reactive group F. The (modified) antibody according to general formula (2) can be prepared by a person skilled in the art using standard organic and / or enzymatic synthesis techniques and as illustrated in the Examples. Antibody AB, linker L 6 , b, x and y are as defined above in the context of the conjugate according to structure (1).

[0447] Reactive part F

[0448] F is reactive to Q in a conjugation reaction defined below, preferably wherein the conjugation reaction is a cycloaddition or a nucleophilic reaction. As will be appreciated by those skilled in the art, the options for F are the same as those for Q, provided that F and Q react with each other. Thus, F preferably comprises a click probe, a sulfhydryl, a sulfhydryl-reactive moiety, an amine or an amine-reactive moiety, more preferably F is a click probe, a sulfhydryl or an amine, most preferably F is a click probe. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene, Sydney ketone, alkene moiety and alkyne moiety. Preferably, the click probe comprises or is azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene or Sydney ketone, most preferably azide. Typical sulfhydryl reactive moieties are selected from maleimide moieties, haloacetamide moieties, malonamide moieties, phosphinamide moieties, cyanoethynyl moieties, o-quinone moieties, vinyl sulfone, vinyl pyridine moieties, or methylsulfonylphenyl oxadiazole moieties. Most preferably, the sulfhydryl reactive moiety comprises or is a maleimide moiety. Typical amine reactive moieties are selected from N-hydroxysuccinimide esters, isocyanates, isothiocyanates, and benzyl halides. In a preferred embodiment, F is a click probe or a sulfhydryl group, more preferably F is an azide or a sulfhydryl group, and most preferably F is an azide.

[0449] More than one reactive group F may be present in an antibody. The reactive group F in an antibody may be naturally present or may be placed in the antibody by specific techniques such as (bio) chemistry or genetic techniques. The reactive group placed in the antibody is prepared by chemical synthesis, such as an azide or a terminal alkyne. Methods for preparing modified antibodies are known in the art, for example, from WO 2014 / 065661, WO 2016 / 170186 and WO 2016 / 053107, which are incorporated herein by reference. From the same literature, the conjugation reaction between the modified antibody and the linker-drug construct is known to those skilled in the art.

[0450] Preferably, F is a click probe reactive toward (hetero)cyclic alkenes and / or (hetero)cyclic alkynes, and is typically selected from the group consisting of azides, tetrazines, triazines, nitrrone, nitrile oxide, nitrile imine, diazo compounds, o-quinone, dioxythiophene, and sydney ketone. Preferred structures of the reactive group are structures (F1)-(F10) depicted below.

[0451]

[0452] Here, the wavy bond represents the connection to the payload. For (F3), (F4), (F8), and (F9), the payload can be connected to any of the wavy bonds. Then another wavy bond can be connected to a selected from hydrogen, C1–C 24 Alkyl groups, C2–C 24 Acyl 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 The R group of the sulfonyl group, each of which (except hydrogen) may be optionally substituted and optionally replaced by one or more selected from O, S and NR 32 The heteroatom interval, where R 32 are independently selected from the group consisting of hydrogen and C1-C4 alkyl. One skilled in the art will understand which R groups are applicable to each group F. For example, the R group attached to the nitrogen atom of (F3) can be selected from alkyl and aryl, and the R group attached to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl. Preferably, the reactive moiety F is selected from an azide or a tetrazine. Most preferably, the reactive moiety F is an azide.

[0453] In a second preferred embodiment, F is a sulfhydryl group or a precursor thereof. The sulfhydryl group or its precursor F is used in a conjugation reaction to link the linker-drug construct to the (modified) antibody. F reacts with the sulfhydryl-reactive probe Q in the sulfhydryl linkage. The sulfhydryl group is preferably a sulfhydryl group of the cysteine ​​amino acid side chain, which is naturally present in the antibody AB, in which case the linker L is not present. 6 (b=0), but it can also optionally be connected via a linker L 6 Thiol precursors in the context of bioconjugation are known in the art and include disulfide bonds, which can be naturally occurring disulfide bonds present in antibodies or synthetically introduced disulfide bonds that are reduced as known in the art. Preferably, F is a thiol group of a cysteine ​​side chain.

[0454] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or its precursor F is used in the conjugation reaction to connect the linker-drug construct to the (modified) antibody. F reacts with the amine-reactive probe Q in a nucleophilic substitution. The amine is typically a primary amine, preferably an amine of the lysine amino acid side chain, which is naturally present in the antibody AB, in which case the linker L is not present. 6 (b=0), but it can also optionally be connected via a linker L 6 Preferably, F is a primary amine group of a lysine side chain.

[0455] Method for synthesizing antibody conjugates according to general structure (1)

[0456] In another aspect, the present invention relates to a method for preparing an antibody conjugate according to the invention, comprising the 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 method occurs under conditions where Q reacts with F to covalently link the antibody AB (3) to a payload D. In the method according to the invention, Q reacts with F to form a covalent link between the antibody and the compound according to the invention. Complementary reactive groups Q and reactive groups F are known to those skilled in the art and are described in more detail below.

[0457] Any conjugation technique known in the art can be used to prepare the multifunctional antibody construct according to the present invention. Suitable conjugation techniques include sulfhydryl linkage, lysine linkage, cycloaddition (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cycloaddition, strain-promoted quinone-alkyne cycloaddition). Preferred conjugation techniques used in the context of the present invention include nucleophilic reaction and cycloaddition, preferably wherein the cycloaddition is [4+2] cycloaddition or [3+2] cycloaddition and the nucleophilic reaction is Michael addition or nucleophilic substitution. Suitable conjugation techniques are disclosed in, for example, GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition 2013 (ISBN: 978-0-12-382239-0), WO 2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233–2242, PCT / EP2021 / 050594, PCT / EP2021 / 050598 and NL2026947.

[0458] Therefore, in a preferred embodiment of the conjugation method according to the present invention, conjugation is accomplished via a nucleophilic reaction such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is acylation of the primary amino group with an activated ester. A preferred Michael reaction is the maleimide-sulfhydryl reaction, which is widely used in bioconjugation.

[0459] Therefore, in a preferred embodiment of the conjugation method according to the present invention, conjugation is completed via cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (such as Diels-Alder reactions) or (3+2)-cycloadditions (such as 1,3-dipolar cycloadditions). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition reaction. Preferred Diels-Alder reactions are inverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, 1,3-dipolar cycloadditions are used, more preferably alkyne-azide cycloadditions, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and those skilled in the art know how to perform them.

[0460] The method according to this aspect preferably involves a click reaction, more preferably a 1,3-dipolar cycloaddition, most preferably an alkyne / azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azide group. Click reactions, such as 1,3-dipolar cycloadditions, are known in the art, and those skilled in the art know how to perform them.

[0461] Thus, according to this aspect, the method for preparing an antibody conjugate according to the invention comprises reacting a modified antibody of structure (3) with a compound according to structure (2) to obtain an antibody conjugate of structure (1).

[0462] In a preferred embodiment, the method for preparing the antibody conjugate according to the present invention comprises:

[0463] (i) an antibody comprising a γ-core N-acetylglucosamine (GlcNAc) portion and a compound of formula S(F) x -P compound in the presence of a catalyst, wherein y = 1, 2, 3 or 4, wherein S (F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The α-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26):

[0464] AB–[GlcNAc(Fuc) w –S{F} x ] y (26)

[0466] in

[0467] -AB is an antibody that can target tumors expressing PTK7;

[0468] -Fuc is fucose;

[0469] -w is 0 or 1; and

[0470] (ii) reacting the modified antibody with a compound according to structure (2):

[0471] Q–L–D (2)

[0473] in:

[0474] -Q is the reactive moiety;

[0475] -L is the linker connecting Z to D;

[0476] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0477] to obtain an antibody conjugate according to structure (1).

[0478] Step (i)

[0479] In step (i), an antibody comprising 1, 2, 3 or 4 core N-acetylglucosamine moieties is combined with an antibody of formula S(F) x -P compound in the presence of a catalyst, wherein S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with a reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The antibody is typically trimmed to core-GlcNAc residues, as further described below. Step (i) provides a modified antibody according to formula (26).

[0480] The starting material, i.e., an antibody comprising a core-GlcNAc substituent, is known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the method according to the present invention further comprises deglycosylation of the antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the polysaccharide, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and / or a combination thereof, the selection of which depends on the nature of the polysaccharide. EndoSH is described in PCT / EP2017 / 052792, see Examples 1 to 3 and SEQ.ID No: 1, which is incorporated herein by reference.

[0481] The structural features S and x are defined above for the antibody conjugates according to the present invention and are also applicable to the present invention. x Connection formula S(F) x Compounds of S(F) are known in the art. For example, Wang et al., Chem. Eur. J. 2010, 16, 13343–13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459–5462, and WO 2009 / 102820, all of which are incorporated herein by reference, disclose a number of compounds S(F)x -P and its synthesis. In a preferred embodiment, S(F) x - The nucleoside monophosphate or diphosphate P in P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP), most preferably P = UDP. Preferably, S(F) x -P is selected from the group consisting of GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetylgalactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-ynoic acid amide)-2-deoxy-galactose-UDP. Most preferably, S(F) x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0482] Able to convert S(F) x Suitable catalysts for the transfer of moieties to the core-GlcNAc moiety are known in the art. Suitable catalysts are catalysts for which the specific sugar derivative nucleotide S(F) in this specific method x-P is a substrate. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group consisting of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group consisting of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcT) and β(1,4)-galactosaminyltransferases (GalT), and most preferably from the group consisting of β(1,4)-N-acetylgalactosaminyltransferases with mutant catalytic domains. Suitable catalysts and mutants thereof are disclosed in WO 2014 / 065661, WO 2016 / 022027, and WO 2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or an N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. Particularly preferred are the mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186. These galactosyltransferase (mutant) enzyme catalysts are able to recognize internal sugars and sugar derivatives as receptors. Thus, the sugar derivative S(F) x In step (i) attachment is made to a core-GlcNAc substituent, whether or not said GlcNAc is fucosylated.

[0483] 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.

[0484] Step (ii)

[0485] In step (ii), the modified antibody is reacted with a compound according to general structure (2) to obtain an antibody conjugate according to structure (1), wherein the compound comprises a reactive group Q capable of reacting with a reactive group F and a payload D, wherein the antibody conjugate contains a linker group Z resulting from the reaction between Q and F. This reaction occurs under conditions in which 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.

[0486] In a preferred embodiment, in step (ii), the azide on the azide-modified antibody reacts with an alkynyl group, preferably a terminal alkynyl group or a (hetero)cycloalkynyl group, of a compound according to general structure (2) via a cycloaddition reaction. This cycloaddition reaction of a molecule comprising an azide with a molecule comprising a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the “click chemistry” reactions known in the art. In the case of a linker-conjugate comprising a terminal alkynyl group, the cycloaddition reaction needs to be carried out in the presence of a suitable catalyst, preferably a Cu(I) catalyst. However, in a preferred embodiment, the linker-conjugate comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl group is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required, and the reaction can even occur spontaneously through a reaction known as strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the “metal-free click chemistry” reactions known in the art.

[0487] application

[0488] The present invention further relates to a method for treating a subject in need thereof, comprising administering an antibody conjugate according to the present invention as defined above. A subject in need 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 conjugates according to the present invention are particularly suitable for this aspect. The described methods are generally suitable for the treatment of cancer. In the method according to this aspect, the antibody conjugate is typically administered in a therapeutically effective dose. This aspect of the invention can also be expressed as an antibody conjugate according to the present invention for treating a subject in need thereof, preferably for treating cancer. In other words, this aspect relates to the use of an antibody conjugate according to the present invention for the preparation of a medicament or pharmaceutical composition for treating a subject in need thereof, preferably for treating cancer. In this article, the treatment of cancer is expected to include treating, imaging, diagnosing, preventing the proliferation of, inhibiting and reducing tumors.

[0489] This aspect of the invention can also be expressed as a method for targeting cells expressing PTK7, particularly PTK7-expressing tumor cells, comprising contacting an antibody conjugate according to the invention with cells that are likely to express PTK7. Thus, the method according to this aspect is suitable for determining whether a cell expresses PTK7. These PTK7-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 are likely to express PTK7 are PTK7-expressing cells. Targeting PTK7-expressing cells preferably includes one or more of the following: treating, imaging, diagnosing, preventing proliferation of, inhibiting, and reducing PTK7-expressing cells, particularly PTK7-expressing tumor cells. The method according to this embodiment can be medical or non-medical. Non-medical methods according to this aspect can involve targeting PTK7-expressing cells in vitro or ex vivo, wherein the cells that are likely to express PTK7 are present in a sample, such as a sample taken from a patient. Such non-medical methods are typically used to diagnose cancer, particularly PTK7-positive cancers.

[0490] In the context of the present invention, the subject may be suffering from a condition selected from the group consisting of colon cancer, lung cancer, breast cancer, ovarian cancer and esophageal cancer. Thus, the treatment of a subject in need thereof preferably refers to the treatment of colon cancer, lung cancer, breast cancer, ovarian cancer and esophageal cancer.

[0491] The inventors surprisingly found that the antibody conjugates according to the present invention are superior to conventional PTK7-targeting antibody conjugates in terms of safety and / or efficacy, such that the therapeutic index of the antibody conjugates according to the present invention is increased relative to conventional PTK7-targeting antibody conjugates.

[0492] Conjugation mode

[0493] In the context of the present invention, "conjugation mode" refers to the method used to conjugate the payload D to the antibody AB, as well as the structural features of the resulting antibody conjugate, in particular the structural features of the linker connecting the payload to the antibody, which are a direct result of the conjugation method. Thus, in one embodiment, the conjugation mode refers to the method by which the payload is conjugated to the antibody. In an alternative embodiment, the conjugation mode refers to the structural features of the linker and / or the point of attachment of the linker to the antibody, which are a direct result of the method by which the payload is conjugated to the antibody.

[0494] In a further aspect, the present invention relates to the use of a conjugation mode for increasing the therapeutic index of an antibody conjugate in the treatment of PTK7-expressing tumors, wherein the conjugation mode is used to link the antibody AB to the payload D via a linker L. The conjugation mode comprises:

[0495] (i) an antibody comprising a γ-core N-acetylglucosamine (GlcNAc) portion and a compound of formula S(F) x -P compounds in the presence of catalyst

[0496] Contact at the bottom, where y = 1, 2, 3 or 4, where S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The α-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26):

[0497] AB–[GlcNAc(Fuc) w –S{F} x ] y (26)

[0499] in

[0500] -AB is an antibody that can target tumors expressing PTK7;

[0501] -Fuc is fucose;

[0502] -w is 0 or 1; and

[0503] (ii) reacting the modified antibody with a compound according to structure (2):

[0504] Q–L–D (2)

[0506] in:

[0507] -Q is the reactive moiety;

[0508] -L is the linker connecting Z to D;

[0509] -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs;

[0510] Obtaining an antibody conjugate according to structure (1)

[0511] Preferably, increasing the therapeutic index of the antibody conjugate is selected from the group consisting of:

[0512] (a) improving the therapeutic efficacy of the antibody conjugate; and / or

[0513] (b) Increasing the tolerability of antibody conjugates.

[0514] The increase in therapeutic efficacy of the antibody conjugates according to the present invention can take the form of a reduction in tumor size and / or a prolonged time to regression when compared to conventional ADCs targeting PTK7. The increase in tolerability of the antibody conjugates according to the present invention can take the form of a reduction in signs of toxicity compared to administration of an ADC targeting PTK7 prepared using conventional techniques. A reduction in symptoms can also be referred to as a reduction in symptoms or side effects of cancer treatment and can involve one or more clinical signs, such as a reduction in weight loss, a reduction in decreased mobility, a reduction in decreased food intake, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology.

[0515] Examples

[0516] General procedures for transient expression and purification of monoclonal antibodies : Evitria (Zurich, Switzerland) transiently expressed various IgGs (cofiluzumab, 4D5, 7C8, or B12) in CHO K1 cells at 1 L, 100 mL, 100 mL, and 5 L scales, respectively. Other IgGs (12C6 and 12C6a) were transiently expressed in ExpiCHO-S(TM) cells (Gibco) at 500 mL scale. Evitria's ProBioGen Non-fucosylated 12C6 was transiently expressed in CHO cells using a HiTrap MabSelect Sure column. 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 25mM Tris pH 7.5, 150mM NaCl (TBS). The retained protein was eluted with 0.1M AcOH pH 2.7. The eluted product was immediately neutralized with 2.5M Tris-HCl pH 8.8 and dialyzed with 20mM histidine, 150mM NaCL, pH 7.5. IgG was then concentrated (>20mg / mL) using a Vivaspin Turbo 15 ultrafiltration device (Sartorius). The sequence of the IgG is given below:

[0517] Cotuzumab (I) light chain (SEQ ID No. 8):

[0518] EIVLTQSPATLSLSPGERATLSCRASESVDSYGKSFMHWYQQKPGQAPRLLIYRASNLES

[0519] GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFI

[0520] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0521] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0522] Catumaxomab (I) heavy chain (SEQ ID No.7):

[0523] QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYAVHWVRQAPGKRLEWIGVISTYNDY

[0524] TYNNQDFKGRVTMTRDTSASTAYMELSRLRSEDTAVYYCARGNSYFYALDYWGQGTS

[0525] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0526] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP

[0527] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

[0528] TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

[0529] QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0530] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0531] 4D5 (II) light chain (SEQ ID No.16):

[0532] EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSR

[0533] FSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPITFGQGTRLEIKRTVAAPSVFIFPPSDE

[0534] QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

[0535] SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0536] 4D5 (II) heavy chain (SEQ ID No.15):

[0537] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAFHWVRQAPGKGLEWVAVISYDGSIK

[0538] YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYYFDYWGQGTLVTVSS

[0539] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0540] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0541] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0542] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

[0543] PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0544] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0545] 7C8 (III) light chain (SEQ ID No.25):

[0546] EIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPAR

[0547] FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPS

[0548] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL

[0549] TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0550] 7C8 (III) Heavy Chain (SEQ ID No. 26):

[0551] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIMDDGS

[0552] NKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGT

[0553] DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

[0554] LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

[0555] KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0556] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0557] AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0558] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0559] 12C6(IV) light chain (SEQ ID No.20):

[0560] EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIP

[0561] DRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPMYT FGQGTKLEIKRTVAAPSVFIFP

[0562] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0563] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0564] 12C6(IV) heavy chain (SEQ ID No.19):

[0565] VQLVQSGGGLVHPGGSLRLSCAGSGFTFS TYLMY WVRQAPGKTLEWVS AIGSGGDTY

[0566] YADSVK GRFTISRDNAKNSLYLQMNSLRAEDMAVYYCAR GLGY WGQGTLVTVSS

[0567] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0568] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0569] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0570] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

[0571] PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0572] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0573] 12C6a(V) light chain (SEQ ID No.22):

[0574] DIQMTQSPSSLSASVGDRVTITC RASQGISSWLA WYQQKPEKAPKSLIY AASSLQS GVP

[0575] SRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSYPYT FGQGTKLEIK

[0576] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

[0577] QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0578] 12C6a(V) heavy chain: See 12C6 heavy chain (SEQ ID No.19) <\

[0579] B12(VI) light chain (SEQ ID No.28):

[0580] EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISD

[0581] RFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPS

[0582] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL

[0583] TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0584] B12(VI) heavy chain (SEQ ID No. 27):

[0585] QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNG

[0586] NKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYY

[0587] MDVWGKGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG

[0588] ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0589] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

[0590] DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0591] KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0592] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0593] General RP-UPLC Procedure (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 minutes. 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 into a Bioresolve RP mAb 2.1*150 mm 2.7 μm column (Waters) at a rate of 0.4 mL / min, with a column temperature of 70°C. A linear gradient from 30% to 54% acetonitrile in 0.1% TFA and water was applied over 9 minutes. The absorbance of the elution peak was measured at 215 nm, followed by automatic integration (MassLynx, Waters) to determine the reaction conversion.

[0594] Mass spectrometry analysis of (modified) monoclonal antibodies General procedure: Prior to mass spectrometry analysis, IgG was treated with IdeS to allow analysis of the Fc / 2 fragment. For analysis of light and heavy chains, a solution of 20 μg (modified) IgG was incubated with 100 mM DTT (total volume 4 μL) at 37°C for 5 minutes. If present, the azide function was reduced to an amine under these conditions. For analysis of the Fc / 2 fragment, a solution of 20 μg (modified) IgG was incubated with IdeS / Fabricator TM The samples were incubated with 1.25 U / μL of phosphate buffered saline (PBS) pH 6.6 (total volume 10 μL) at 37°C for 1 hour. The samples were diluted to 80 μL and then subjected to electrospray ionization time-of-flight (ESI-TOF) analysis on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0595] General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg / mL) was mixed with 1% w / w EndoSH (as described in PCT / EP2017 / 052792, see Examples 1-3, and SEQ. ID No: 1, which are incorporated herein by reference), 3% w / w His-TnGalNAcT (as described in PCT / EP2016 / 059194, see Examples 3 and 4, and SEQ. ID No: 49, which are incorporated herein by reference), 0.01% AP (Roche), and UDP 6-N3-GalNAc ( Figure 3Compound 2d in (25eq compared to IgG) was incubated together at 30°C for 16 hours in 6mM MnCl2 and TBS. Next, functionalized IgG was purified using a HiTrapMabSelect sure 5mL column. After loading the reaction mixture, the column was washed with TBS+0.2% triton and TBS. IgG was eluted with 0.1M AcOH pH 2.7 and neutralized with 2.5M Tris-HCl pH 8.8. After dialysis 3 times with 20mM histidine, 150mM NaCl pH 7.5, IgG was concentrated to 15-20mg / mL using a Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0596] Preparation of Azide-Functionalized Antibodies: Examples 1–7:

[0597] Example 1: Preparation of Cofiltuzumab-(6-N3-GalNAc)2(I-N3)

[0598] According to the general procedure of enzymatic remodeling, cofetiuzumab was converted to cofetiuzumab-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product (observed mass 24329 Da, approximately 90% of the total Fc / 2), which corresponded to the expected product.

[0599] Example 2: Preparation of B12-(6-N3-GalNAc)2(VI-N3)

[0600] According to the general procedure of enzymatic remodeling, B12 was converted to B12-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product (observed mass 24330 Da, accounting for approximately 90% of the total Fc / 2), which corresponded to the expected product.

[0601] Example 3: Preparation of 4D5-(6-N3-GalNAc)2(II-N3)

[0602] According to the general procedure of enzymatic remodeling, 4D5 was converted to 4D5-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, approximately 90% of total Fc / 2) and a minor Fc / 2 product (observed mass 24186 Da, approximately 10% of total Fc / 2), corresponding to the non-fucosylated product.

[0603] Example 4: Preparation of 7C8-(6-N3-GalNAc)2(III-N3)

[0604] According to the general procedure of enzymatic remodeling, 7C8 was converted to 7C8-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, approximately 90% of total Fc / 2) and a minor Fc / 2 product (observed mass 24186 Da, approximately 10% of total Fc / 2), corresponding to the non-fucosylated product.

[0605] Example 5: Preparation of 12C6-(6-N3-GalNAc)2(IV-N3)

[0606] According to the general procedure of enzymatic remodeling, 12C6 was converted to 12C6-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product corresponding to the expected product (observed mass 24331 Da, approximately 90% of total Fc / 2) and a minor Fc / 2 product (observed mass 24186 Da, approximately 10% of total Fc / 2), corresponding to the non-fucosylated product.

[0607] Example 6: Preparation of 12C6a-(6-N3-GalNAc)2(V-N3)

[0608] According to the general procedure of enzymatic remodeling, 12C6a was converted to 12C6a-(6-N3-GalNAc)2. Mass spectrometry analysis of the sample after IdeS treatment showed a major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, approximately 90% of total Fc / 2) and a minor Fc / 2 product (observed mass 24186 Da, approximately 10% of total Fc / 2), corresponding to the non-fucosylated product.

[0609] Example 7: Preparation of aFuc-12C6-(6-N3-GalNAc)2 (aFuc-IV-N3)

[0610] Following the general procedure for enzymatic remodeling, aFuc-12C6 was converted to aFuc-12C6-(6-N3-GalNAc)2. Mass spectrometry analysis of the IdeS-treated sample revealed a major Fc / 2 product (observed mass 24187 Da, approximately 95% of total Fc / 2), corresponding to the expected product.

[0611] Examples 8-12: Synthesis of Linker Conjugates 3, 4, 5b and 9

[0612]

[0613] Example 8: Preparation of Compound 11

[0614] Compound 10 (163 mg, 240 μmol) was added to a mixture of exitecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in anhydrous DMF (0.9 mL). After 20 h, the reaction mixture was diluted to 9 mL DCM and purified by gradient column chromatography (0 → 40% MeOH / DCM) to give 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) calculated value C 55 H 54 FN6O 10 + (M+H) + 977.39, found 977.72. In addition to 11, ixitecan free base (82.4 mg, 189 μmol, 20%) was also recovered. LCMS (ESI+) calculated value C 24 H 23 FN3O4 + (M+H) + 436.46, measured value 436.54.

[0615] Example 9. Preparation of Compound 3

[0616] The synthesis of BCN-HS-(va-PABC-Ex)2(3) is also described in PCT / EP2021 / 075401 (Example 4), which is incorporated herein. To a solution of compound 11 (155 mg, 159 μmol) in DMF (1.6 mL) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 12 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL) and purified by gradient column chromatography (0→40% MeOH / DCM) to give 3 as a light yellow solid (94 mg, 44 μmol, 28%). 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + LCMS (ESI+) calcd. 1066.88, found 1067.12.

[0617]

[0618] Example 10: Preparation of Linker-Conjugate 4

[0619] The synthesis of BCN-HS-(vc-PABC-MMAE)2(4) is also described in PCT / EP2017 / 052791 (Example 25), which is incorporated herein. To a solution of 14 (27 mg, 33 μmol) in DMF (400 μL) was added a solution of triethylamine (22 μL; 16 mg; 158 μmol) and vc-PABC-MMAE.TFA 13 (96 mg; 78 μmol) in DMF (1.0 mL). The mixture was allowed to stand for 19 h, and then 2,2'-(ethylenedioxy)bis(ethylamine) (37 μL, 38 mg, 253 μmol) was added. After 2 h, the reaction mixture was diluted with DMF (100 μL) and purified by RP HPLC (C18, 30%→90% MeCN (1% AcOH) / water (1% AcOH)). The desired product 4 (41 mg, 14.7 μmol, 45%) was obtained as a colorless film. LCMS (ESI + ) Calculated value C 138 H 219 N 23 O 35 S 2+ (M+2H + )1395.79 Actual measured value 1396.31.

[0620] Example 11: Synthesis of Linker Conjugate 5b

[0621] The synthesis of BCN-HS-vc-PABC-CM (5b) is prepared according to the procedure described in WO2019110725 incorporated herein. To a solution of 15 (9.0 mg, 4.88 μmol, compound 138 in WO2019110725) in DMF (445 μL), a solution of 16 (12.8 mg, 24.4 μmol, compound 3 in WO2019110725) in DMF (45.8 μL) and 50% v / v Et3N solution (8.98 μL, 32.2 μmol) in DMF was added. The mixture was allowed to stand for 4 h, then diluted with DCM to a cumulative volume of 4.5 mL. The mixture was purified by silica gel chromatography (0 → 20% MeOH in DCM solution). The desired product 5b (5.0 mg, 2.24 μmol, 46%) was obtained as a colorless oil. LCMS (ESI+) calculated value C 96 H 136 IN9O 35 S4 2+ (M+2H + )1115.35, measured value 1115.40.

[0622]

[0623] Example 12. Preparation of Compound 9

[0624] A solution of BCN-HS-PEG2-b-(Glu(OFm)-OH)2 (8, 12.1 mg, 10 μmol, 1.0 eq) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-isotecan (5b, Fmoc deprotection 5, 19 mg, 25 μmol, 2.5 eq) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 eq) and HATU (8.9 mg, 23 μmol, 2.3 eq). After stirring at room temperature for 2 h, the reaction mixture was further diluted with DCM (800 μL) and purified by silica gel flash column chromatography (0% → 20% MeOH / DCM) to give a clear oily product (the yield was difficult to determine due to the presence of DMF). LCMS (ESI+) calculated value C 140 H 150 F2N 17 O 33 S + (M / 2+H + )1334.01, measured value 1334.79.

[0625] The compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 eq) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel flash column chromatography (0% → 45% MeOH / DCM) to afford compound 9 in 44% yield as a yellow solid (10.2 mg, 4.4 μL). LCMS (ESI+) calculated value C 112 H 130 F2N 17 O 33 S + (M / 2+H + )1156.2, measured value 1156.74.

[0626] Examples 13-24: Conjugation of linker payloads to (modified) monoclonal antibodies

[0627] Example 13: Production of Cofetiuzumab-Aur0101

[0628] Cotuzumab (14.4 mg, 4.34 mg / ml, dissolved in 20 mM histidine, 6% sucrose) was added with 5% pH adjustment buffer (0.5 M Tris, 25 mM EDTA, pH 8.5) and 2.3 molar equivalents of TCEP (1 mM in water) and incubated at room temperature for 2 hours. The reduced antibody was added to DMA to obtain 10% v / v and 6 molar equivalents of auristatin 0101 (2 mM in DMA) and incubated at room temperature for 1 hour. The reaction was quenched with 6 molar equivalents of NAC (100 mM in water). The conjugate was purified by preparative discontinuous diafiltration into 20 mM histidine / 6% sucrose in Vivaspins. 0.04% Tween-20 was added before filter sterilization. The average DAR was measured to be 4.3.

[0629] Example 14: Conjugation of Cofetuzumab (6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain the conjugate Cofetuzumab-3

[0630] To colfetuzumab (6-N3-GalNAc) 2 solution (1664 μ L, 32.0 mg, 19.37 mg / ml, in TBS, pH 7.5), sodium deoxycholate (322 μ L, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex) 2 3 (86 μ L, 10 mM DMF solution) and propylene glycol (881 μ L) were added. The reactants were incubated at room temperature overnight. Next, the conjugate was purified on the HiLoad 16 / 600 Superdex200 PG column (Cytiva) on AKTA Pure (Cytiva). In order to remove excess free payload, 25 mg activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. Charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μ m, Corning). The solution was then buffer-exchanged using a HiTrap26-10 desalting column (Cytiva), washed with 0.1 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer, pH 6.0, supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of samples after IdeS treatment revealed a major Fc / 2 product (observed mass 26,461 Da, approximately 90% of total Fc / 2), corresponding to the conjugate cofetizumab-3. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.86.

[0631] Example 15: Conjugation of Cofetuzumab (6-N3-GalNAc) 2 with BCN-HS-(vc-PABC-MMAE) 2 4 to obtain the conjugate Cofetuzumab-4

[0632] To colfetuzumab (6-N3-GalNAc) 2 (463 μ L, 9.0 mg, 19.37 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (60 μ L, 110 mM) and BCN-HS- (vc-PABC-MMAE) 2 4 (1.8 μ L, 100 mM DMF solution) and DMF (58 μ L) were added. The reactants were incubated at room temperature overnight. Next, the conjugate was purified on a HiLoad 10 / 300 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). The solution was subsequently buffer-exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20 was added before filtration sterilization. Mass spectrometry analysis of samples after IdeS treatment showed a major Fc / 2 product (observed mass 27119 Da, approximately 90% of total Fc / 2), corresponding to the conjugate cofilotuzumab-4. RP-UPLC analysis of samples under reducing conditions showed an average DAR of 3.90.

[0633] Example 16: Conjugation of Cofetuzumab (6-N3-GalNAc) 2 with BCN-HS-vc-PABC-calicheamicin 5b to obtain the conjugate Cofetuzumab-5b

[0634] To cofetiuzumab (6-N3-GalNAc) 2 (730 μL, 14.0 mg, 19.37 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (94 μL, 110 mM) and BCN-HS-vc-PABC-calicheamicin 5b (7 μL, 40 mM DMF solution) and DMF (87 μL) were added. The reactants were incubated at room temperature overnight. Next, the conjugate was purified on a HiLoad 10 / 300 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). The solution was subsequently buffer-exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20 was added before filter sterilization. Mass spectrometry analysis of samples after IdeS treatment showed a major Fc / 2 product (observed mass 26714 Da, approximately 90% of total Fc / 2), corresponding to the conjugate cofilotuzumab-5b. RP-UPLC analysis of samples under reducing conditions showed an average DAR of 1.93.

[0635] Example 17: Conjugation of B12(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate B12-3

[0636] To B12(6-N3-GalNAc)2(6.33mL, 150.0mg, 23.71mg / ml, in TBS, pH 7.5) solution, add BCN-HS-PEG2-HS-(va-PAB-Ex)23(495 μ L, 10mM DMF solution) and propylene glycol (7.0mL).The reactant is incubated at room temperature overnight.Next, the conjugate is purified on HiLoad 26 / 600Superdex200 PG column (Cytiva) on AKTA Pure (Cytiva).In order to remove excessive free payload, 150mg activated carbon (Carbon RHC, Filtrox AG) is added and rotated overnight.Charcoal is removed by centrifugation and subsequently filtered through PES syringe filter (pore size 0.20 μm, Corning).The solution is then dialyzed to 20mM histidine, 6% sucrose buffer pH 6.0 2 hours at room temperature and overnight at 4 ° C. The solution was concentrated using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius), and 0.04% Tween-20 was added before filter sterilization. Mass spectrometry analysis of samples after IdeS treatment revealed a major Fc / 2 product (observed mass 26,462 Da, approximately 90% of the total Fc / 2), corresponding to conjugate B12-3. RP-UPLC analysis of the samples under reducing conditions revealed an average DAR of 3.75.

[0637] Example 18: Conjugation of 4D5(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 4D5-3

[0638] To 4D5 (6-N3-GalNAc) 2 (424 μ L, 8.8 mg, 20.74 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (88 μ L, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex) 2 3 (23.4 μ L, 10 mM DMF solution) and propylene glycol (240 μ L) were added. The reactants were incubated at room temperature overnight. The conjugate was then purified using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with PBS pH 7.4 on an AKTA Pure (Cytiva). In order to remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μ m, Corning). The solution was then dialyzed three times into 20 mM histidine, 6% sucrose buffer, pH 6.0, supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of samples treated with IdeS revealed a major Fc / 2 product (observed mass 26,465 Da, approximately 90% of total Fc / 2) corresponding to conjugate 4D5-3. A minor Fc / 2 product (observed mass 26,318 Da, approximately 10% of total Fc / 2) corresponded to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.91.

[0639] Example 19: Conjugation of 7C8(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 7C8-3

[0640] To 7C8(6-N3-GalNAc)2(313 μL, 6.9 mg, 22.14 mg / ml, in TBS, pH 7.5) solution, BCN-HS-PEG2-HS-(va-PAB-Ex)23(23.1 μL, 10 mM DMF solution) and propylene glycol (254 μL) were added. The reactants were incubated at room temperature overnight. Next, the conjugate was purified on a Superdex20010 / 300 incremental column (Cytiva) on an AKTA Pure (Cytiva). In order to remove excess free payload, 11 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then buffer-exchanged using a HiTrap 26-10 desalting column (Cytiva), washed with 0.1 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer, pH 6.0, and supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of samples after IdeS treatment revealed a major Fc / 2 product (observed mass 26,466 Da, approximately 90% of total Fc / 2), corresponding to conjugate 7C8-3. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.95.

[0641] Example 20: Conjugation of 12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 12C6-3

[0642] To 12C6(6-N3-GalNAc)2(481 μL, 10.1 mg, 21.01 mg / ml, in TBS, pH 7.5) solution, BCN-HS-PEG2-HS-(va-PAB-Ex)23(33.7 μL, 10 mM DMF solution) and propylene glycol (370 μL) were added. The reactants were incubated at room temperature overnight. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and balanced with PBS pH 7.4 on an AKTA Pure (Cytiva). In order to remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer, pH 6.0, and supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment revealed a major Fc / 2 product (observed mass 26,466 Da, approximately 95% of total Fc / 2), corresponding to the conjugate 12C6-3. A minor Fc / 2 product (observed mass 26,318 Da, approximately 5% of total Fc / 2) corresponded to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.85. Example 21: Conjugation of 12C6a(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain the conjugate 12C6a-3.

[0643] To 12C6a (6-N3-GalNAc) 2 (484 μL, 10.9 mg, 22.53 mg / ml, in TBS, pH 7.5) solution, BCN-HS-PEG2-HS- (va-PAB-Ex) 2 3 (36.3 μL, 10 mM DMF solution) and propylene glycol (400 μL) were added. The reactants were incubated at room temperature overnight. Next, the conjugate was purified on a Superdex20010 / 300 incremental column (Cytiva) on an AKTA Pure (Cytiva). In order to remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then dialyzed three times into 20 mM histidine, 6% sucrose buffer, pH 6.0, supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of samples after IdeS treatment revealed a major Fc / 2 product (observed mass 26,466 Da, approximately 95% of total Fc / 2), corresponding to the conjugate 12C6a-3. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.90.

[0644] Example 22: Conjugation of 12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-(eva-PAB-Ex)29 to obtain conjugate 12C6-9

[0645] To 12C6(6-N3-GalNAc)2(340 μL, 10 mg, 29.45 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (100 μL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)29 (20 μL, 10 mM DMF solution) and propylene glycol (280 μL) were added. The reactants were incubated at room temperature overnight. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and balanced with PBS pH 7.4 on an AKTA Pure (Cytiva). In order to remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added and rotated overnight. Charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (pore size 0.20 μm, Corning). The solution was then buffer-exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer, pH 6.0, supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of the IdeS-treated sample revealed a major Fc / 2 product (observed mass 26,466 Da, approximately 95% of the total Fc / 2) corresponding to the conjugate 12C6-9. A minor Fc / 2 product (observed mass 26,643 Da, approximately 5% of the total Fc / 2) corresponded to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.84.

[0646] Example 23: Conjugation of aFuc-12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain the conjugate aFuc-12C6-3

[0647] To aFuc-12C6(6-N3-GalNAc)2(549 μL, 13 mg, 23.69 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (130 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (34.7 μL, 10 mM DMF solution) and propylene glycol (355 μL) were added. The reactants were incubated at room temperature overnight. The conjugate was then purified using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and balanced on an AKTA Pure (Cytiva) using PBS pH 7.4. The solution was then buffer-exchanged using a HiTrap 26-10 desalting column (Cytiva), washed with 0.2 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer, pH 6.0, and supplemented with 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment revealed a major Fc / 2 product (observed mass 26,318 Da, approximately 95% of the total Fc / 2), corresponding to the conjugate aFuc-12C6-3. RP-UPLC analysis of the sample under reducing conditions revealed an average DAR of 3.74.

[0648] Example 24: Conjugation of aFuc-12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-(eva-PAB-Ex)29 to obtain the conjugate aFuc-12C6-9

[0649] To aFuc-12C6(6-N3-GalNAc)2 (59 μL, 1.4 mg, 23.69 mg / ml, in TBS, pH 7.5) solution, sodium deoxycholate (14 μL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)2 9 (2.8 μL, 10 mM DMF solution) and propylene glycol (39 μL) were added. The reactants were incubated overnight at room temperature. The conjugate was then purified using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and balanced with PBS pH 7.4 on an AKTA Pure (Cytiva). RP-UPLC analysis of the conjugate aFuc-12C6-9 sample under reducing conditions showed an average DAR of 3.81.

[0650] Examples 25–28: In vitro assays

[0651] Example 25. Binding Assays of hPTK7, cPTK7, and rPTK7 to mAbs and ADCs Using ELISA

[0652] Nickel NTA Plate (Pierce TMNickel plating, ThermoScientific TM ) were washed three times before use. Human PTK7 (CCK4 protein, ECD, His tag, Sino Biological), cynomolgus monkey PTK7 (CCK4 protein, ECD, His tag, Sino Biological), and rat PTK7 (His tag, Acro Biosystems) were dissolved in 0.1% BSA in PBS (PBA) at a concentration of 0.05 μg / mL. 100 μL was added to each well and incubated with shaking at room temperature for 1 hour. After removal, the plate was washed three times with 0.05% Tween-20 in PBS (wash buffer). ADC was diluted to a final concentration of 8 μg / mL in 0.1% PBA, and 100 μL was added to each well (in triplicate). ADC was incubated at room temperature for 1 hour. The plate was washed three times with wash buffer before adding 100 μL of a 1:1000 diluted secondary antibody (goat anti-human IgG, HRP conjugate, Invitrogen). The plate was incubated again at room temperature for 1 h, then washed 3 times with wash buffer and 3 times with PBS. Finally, 100 μL of TMB ELISA substrate (1Step TM TurboTMB ELISA substrate, ThermoScientific TM ) and incubate for 1 minute. The absorbance of the colorimetric signal was measured at 652 nm using a Tecan M1000. The data plotted were background corrected (see Figure 8 A, 8B and Figure 11 ).

[0653] Example 26. Human and mouse serum stability

[0654] The stability of ADC was tested in mouse and human plasma. Protein A agarose (1 mL agarose / mL serum) was used to deplete all IgG from the plasma. ADC was added to the depleted human / mouse serum to a final concentration of 0.1 mg / mL and then incubated at 37°C. At each time point, 0.5 mL was quickly frozen and stored at -80°C until further analysis. To isolate ADC after incubation, 20 μl Protein A agarose resin was added to the sample and incubated at room temperature for 1 hour. The resin was washed 3 times with PBS, followed by addition of 0.1 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). The PBS sample was spin filtered 3 times using an Amicon Ultra spin filter 0.5 mL MWCO 10 kDa (Merck Millipore) and the volume was reduced to 40 μL, resulting in a final ADC concentration of approximately 1 mg / mL. The samples were analyzed on RP-UPLC (DTT reduced) to determine the DAR, as shown in the following table.

[0655] Table 1. Human plasma:

[0656]

[0657] Table 2. Mouse plasma:

[0658]

[0659] - Signal is too weak

[0660] *Measured directly (not by separation from plasma using protA agarose resin).

[0661] The brackets are the percentage of t=0

[0662] Example 27: In vitro cytotoxicity

[0663] A431 (PTK7+, ATCC CRL-1555) cells were seeded in 96-well plates (10,000 cells in 150 μL / well) in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and incubated overnight at 37°C and 5% CO2 in a humidified atmosphere. ADC was added in triplicate as a square root of 10 dilution series to obtain a final concentration in the range of 100 nM–0.01 nM. The cells were incubated for 5 days at 37°C and 5% CO2 in a humidified atmosphere. The culture medium was replaced with 0.01 mg / mL resazurin (Sigma Aldrich) in DMEM supplemented with 10% FBS (200 μL / well). After approximately 4 hours at 37°C and 5% CO2 in a humidified atmosphere, the cells were analyzed using a fluorescence plate reader ( Fluorescence was detected at 560 nm excitation and 590 nm emission using a Tecan M1000. Relative fluorescence units (RFU) were normalized to percentage cell viability by setting wells without cells as 0% viability and wells containing untreated cells as 100% viability (see Figure 9The IC values ​​of ADCs against A431 cells were calculated by nonlinear regression using Graphpad Prism software. 50 values, and are shown in the following table:

[0664] Table 3. IC of various ADCs and mAbs in A431 cells 50 value

[0665] <![CDATA[IC 50 (nM) <!-- 61 -->]]> Cotuzumab-4 3.9 Cotuzumab-5b 0.10 B12-3 >100

[0666] Example 28. Observation of the binding affinity of 12C6-3 and 12C6 naked mAb to FcγR using Biacore

[0667] The binding of the test antibodies to high- and low-affinity human Fcγ receptors was assessed by single-cycle analysis using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software (Cytiva, Uppsala, Sweden) at a flow rate of 30 μl / min. Human Fc receptors FcγRI, FcγRIIA (polymorphisms 167R and 167H), FcγRIIB, FcγRIIIA (polymorphisms 176F and 176V), and FcγRIIIB were obtained from Sino Biological (Beijing, China). FcγRs were captured on a CM5 sensor chip pre-coupled using standard amine chemistry using a His capture kit (Cytiva, Uppsala, Sweden).

[0668] At the beginning of each cycle, His-tagged Fcγ receptors (ligands) diluted in HBS-P+ (Cytiva, Uppsala, Sweden) were loaded to a specific RU level. For each receptor tested, a five-point, three-fold dilution range of the test antibody (analyte) was used without regeneration between each concentration. The test antibody was passed through the chip at 30 μl / min with increasing concentrations, followed by a single dissociation step. After dissociation, the chip was regenerated by injecting glycine at pH 1.5. The signal from the reference channel Fc1 (blank) was subtracted from the signal of the Fc loaded with the receptor to correct for the difference in nonspecific binding to the reference surface. A 1:1 kinetic analysis was performed on the sensorgram of the high-affinity Fcγ receptor hFcγRI, and a steady-state binding analysis was performed on the low-affinity Fcγ receptor.

[0669] Table 4. Summary of the binding of the tested antibodies and ADCs to different human Fcγ receptors. The relative binding range (KD) is expressed as: +++++, 10 -9 to 10 -10 M range;++++,10 -8 M range: +++, 10 -7 M range;++,10 -6 M,+,10-5 The M range was determined by Biacore.

[0670]

[0671] Examples 29–34: CDX Evaluation and In Vivo Studies

[0672] Example 29: IHC staining of PTK7 by NCI-H446 (performed at Crown Biotech, Taicang, China)

[0673] Formalin-fixed paraffin-embedded (FFPE) blocks were sliced ​​onto slides at a thickness of 4 μm on a manual rotary microtome. The slides were baked at 60°C for 30 min. A Bond RX automatic stainer (Leica) was used for staining according to the following procedure: the slides were first treated with a dewaxing solution (Leica) at 72°C for 0.5 min, then treated at room temperature and moved to alcohol, and treated 3 times at room temperature. The slides were then washed 3 times with Bond wash buffer (Leica). After a quick rinse in the same solution, epitope retrieval was performed in Bond ER2 solution (Leica) at 100°C for 20 min. The slides were then briefly washed four times in Bond wash buffer and then washed at room temperature for 3 minutes. Peroxide blocking was performed at room temperature for 10 minutes, followed by washing again 3 times. Subsequently, rabbit anti-human PTK7 primary antibody (PA5-82070, Thermo Fisher, 0.25 μg / mL) was added and incubated at room temperature for 1 hour. The slides were then washed quickly once and three times for 2 minutes in Bond wash buffer. A secondary antibody, goat anti-rabbit-HRP (Leica, ready-to-use), was added and incubated at room temperature for 20 minutes, followed by one quick wash and three two-minute washes in Bond wash buffer. The slides were rinsed with deionized water and then incubated with 3,3'-diaminobenzidine (DAB) Refine for 5 minutes at room temperature. After three washes in deionized water, the slides were counterstained with hematoxylin for 10 minutes. Finally, all slides were washed with deionized water and Bond wash buffer.

[0674] All stained sections were scanned at 40× magnification using the NanoZoomer-HT 2.0 imaging system. All images of non-necrotic tumor areas were acquired using the HALO TM The analysis was performed using the ELISA platform. The intensity of membrane positive staining was divided into four levels: 0 (negative), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). The H score was used to assess the percentage of cells at different intensity levels. H score = (% of 0 level) × 0 + (% of 1 level) × 1 + (% of 2 level) × 2 + (% of 3 level) × 3 (H score ranges from 0 to 300). The H score of the NCI-H446 lung cancer xenograft cell line is shown in Table 4.

[0675] Table 4. IHC score NCI-H446

[0676]

[0677] Example 30: In vivo efficacy study (conducted at Crown Biotechnology Co., Ltd., Zhongshan, China)

[0678] NCI-H446 is a lung cancer xenograft model cell line, cultured in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with a standard 5% CO2 environment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0679] 7-9 week old BALB / c nude mice (female) were injected subcutaneously in the right anterior flank with 5×10 6 When the average tumor size reaches 100-200mm 3 At 14:00, mice were randomly divided into 10 groups of 8 mice each and treatment was started. Randomization was based on the "matched distribution" method (Study Director TM Software, version 3.1.399.19). The date of randomization will be denoted as day 0.

[0680] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice a week. The experimental endpoint was tumor volume reaching 3,000 mm 3 , weight loss of more than 20% or 28 days (whichever comes first).

[0681] Randomization: When the mean tumor size reaches approximately 100-200 mm 3 Randomization was performed at the time of the NCI-H446 model study. A total of 80 mice were included in the NCI-H446 model study and randomly divided into 10 groups of 8 mice each. TM Randomization was performed using the BIOMEDICAL® software, version 3.1.399.19. The date of randomization will be denoted as day 0.

[0682] Test article administration: Test article administration was performed via intravenous injection into the tail vein at a volume of 10 mL / kg. Treatment began on the same day as randomization. Administration was performed in a laminar flow cabinet.

[0683] Observation and data collection.After tumor cell inoculation, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for any adverse effects of tumor growth and treatment on normal behavior, such as activity, visual estimation of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume was measured in two dimensions using a caliper every 3-4 days, and volume data was expressed in mm3 using the formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Dose, tumor, and body weight measurements will be performed in a laminar flow cabinet.

[0684] Table 5. Summary of test items and dose levels included in the in vivo studies

[0685]

[0686] Tumor volume and mouse body weight over time in efficacy studies using the above test items are shown in Figure 2. Figure 10 As shown in A–10D.

[0687] Example 31: In vivo efficacy study

[0688] NCI-H446 is a lung cancer xenograft model cell line, cultured in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with a standard 5% CO2 environment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0689] 7-9 week old BALB / c nude mice (female) were injected subcutaneously in the right anterior flank with 5×10 6 When the average tumor size reaches 100-200mm 3 At the same time, the mice were randomly divided into 6 groups with 8 mice in each group and the treatment was started. Randomization was based on the "matched distribution" method (Study Director TM Software, version 3.1.399.19). The date of randomization will be denoted as day 0.

[0690] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice a week. The experimental endpoint was tumor volume reaching 3,000 mm 3 , weight loss of more than 20% or 77 days (whichever comes first).

[0691] Randomization: When the mean tumor size reached approximately 136 mm 3Randomization was performed at the time of the NCI-H446 model study. A total of 48 mice were included in the NCI-H446 model study and randomly divided into 6 groups of 8 mice each. TM Randomization was performed using the BIOMEDICAL® software, version 3.1.399.19. The date of randomization will be denoted as day 0.

[0692] Test article administration: Test article administration was performed via intravenous injection into the tail vein at a volume of 10 mL / kg. Treatment began on the same day as randomization. Administration was performed in a laminar flow cabinet.

[0693] Observation and data collection : After tumor cell inoculation, animals are checked daily for morbidity and mortality. During routine monitoring, animals are checked for any adverse effects of tumor growth and treatment on normal behavior, such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume is measured in two dimensions using calipers every 3-4 days, and volume data is expressed in mm3 using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Dose, tumor, and body weight measurements will be performed in a laminar flow cabinet.

[0694] Table 6. Summary of test items and dose levels included in the in vivo studies

[0695]

[0696] Tumor volume and mouse body weight over time in the efficacy study with the above test items and the corresponding Kaplan-Meier plots are depicted in Figure 12A , 12B and 12C.

[0697] Example 32: In vivo efficacy study

[0698] 7-9 week old female BALB / c nude mice received tumor fragments of BR1282, a breast cancer patient-derived xenograft model cell line. Tumor fragments (2-3 mm in diameter) were inoculated subcutaneously in the right upper flank region. When the average tumor size reached 100-200 mm 3 At the time of treatment, mice were randomly divided into 9 groups of 8 mice each and the treatment was started. Randomization was based on the "matched distribution" method (Study Director TM Software, version 3.1.399.19). The date of randomization will be denoted as day 0.

[0699] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice a week. The experimental endpoint was tumor volume reaching 3,000 mm 3 , weight loss of more than 20% or 39 days (whichever comes first).

[0700] Randomization: When the mean tumor size reaches approximately 135 mm 3 A total of 72 mice were included in the BR1282 model study and randomly divided into 9 groups of 8 mice each. TM Randomization was performed using the BIOMEDICAL® software, version 3.1.399.19. The date of randomization will be denoted as day 0.

[0701] Test article administration: Test article administration was performed via intravenous injection into the tail vein at a volume of 10 mL / kg. Treatment began on the same day as randomization. Administration was performed in a laminar flow cabinet.

[0702] Observation and data collection : After tumor cell inoculation, animals are checked daily for morbidity and mortality. During routine monitoring, animals are checked for any adverse effects of tumor growth and treatment on normal behavior, such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume is measured in two dimensions using calipers every 3-4 days, and volume data is expressed in mm3 using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Dose, tumor, and body weight measurements will be performed in a laminar flow cabinet.

[0703] Table 7. Summary of test items and dose levels included in the in vivo studies

[0704]

[0705] Tumor volume and mouse body weight over time in the efficacy study with the above test items are depicted in Figure 13A and 13B.

[0706] Example 33: In vivo efficacy study

[0707] NCI-H446 is a lung cancer xenograft model cell line, cultured in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with a standard 5% CO2 environment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0708] 7-9 week old BALB / c nude mice (female) were injected subcutaneously in the right anterior flank with 5×10 6 When the average tumor size reaches 100-200mm 3 At the time of treatment, mice were randomly divided into 5 groups of 8 mice each and the treatment was started. Randomization was based on the "matched distribution" method (Study Director TM Software, version 3.1.399.19). The date of randomization will be denoted as day 0.

[0709] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice a week. The experimental endpoint was tumor volume reaching 3,000 mm 3 , weight loss of more than 20% or 28 days (whichever comes first).

[0710] Randomization: When the mean tumor size reaches approximately 100-200 mm 3 Randomization was performed at the time of the NCI-H446 model study. A total of 40 mice were included in the NCI-H446 model study and randomly divided into 5 groups of 8 mice each. TM Randomization was performed using the BIOMEDICAL® software, version 3.1.399.19. The date of randomization will be denoted as day 0.

[0711] Test article administration: ADC test article administration was performed via intravenous injection in the tail vein at a volume of 10 mL / kg. For ADC, only a single dose was administered, starting on the same day as randomization. Olaparib was administered orally at a dose of 50 mg / kg QD for 21 days, also starting on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0712] Observation and data collection : After tumor cell inoculation, animals are checked daily for morbidity and mortality. During routine monitoring, animals are checked for any adverse effects of tumor growth and treatment on normal behavior, such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormal effects. Tumor volume is measured in two dimensions using calipers every 3-4 days, and volume data is expressed in mm3 using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Dose, tumor, and body weight measurements will be performed in a laminar flow cabinet.

[0713] Table 8. Summary of test items and dose levels included in the in vivo studies

[0714]

[0715] Tumor volume and mouse body weight over time in the efficacy study with the above test items are depicted in Figure 14A and 14B.

[0716] Example 34: In vivo tolerance study

[0717] Sprague Dawley rats (female), 6-7 weeks old, were acclimated for one week before participation in the study.

[0718] Randomization: In the tolerance study, a total of 15 rats were enrolled and randomly divided into 5 study groups, with 3 rats in each group. Randomization was performed based on the "matched distribution" method (StudyDirector™ software, version 3.1.399.19). The randomization date is denoted as day 0.

[0719] Test article administration: Treatment began on the same day of randomization (Day 0) for each study design. The dose levels selected were 75, 100, 125, and 150 mg / kg, single dose. All animals were dosed via slow intravenous injection (10 mL / kg).

[0720] Post-randomization observations and data collection: Animals were examined daily for morbidity and mortality. During routine monitoring, any effects of treatment on animal behavior, such as activity, food and water consumption, weight gain / loss, eye / hair matting, and any other abnormalities, were examined. Detailed records of mortality and observed clinical signs were kept for individual animals. Dosing and body weight measurements were performed in a laminar flow cabinet. Body weight was measured daily using StudyDirector™ software (version 3.1.399.19).

[0721] exist Figure 15 Figure 2 depicts the changes in rat body weight over time at multiple dose levels using 12C6-3 as the test item.

Claims

1. An antibody conjugate according to the general structure (1): AB–[(L 6 ) b –{Z–L–D} x ] y (1) in: -AB is an antibody that can target tumors expressing PTK7; -L is the linker connecting Z to D; -Z is a linking group; -L 6 Yes – GlcNAc(Fuc) w –(G) j –S–(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is –N(H)C(O)CH2–, –N(H)C(O)CF2– or –CH2–; -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs; -b is 0 or 1; -x is 1 or 2; and -y is 1, 2, 3, or 4.

2. The antibody conjugate of claim 1, wherein D is selected from the group consisting of anthracyclines, camptothecins, maytansinoids, enediynes, auristatins, and pyrrolobenzodiazepines. The group consisting of dimers, preferably wherein: -D is an enediyne, preferably selected from the group consisting of calicheamicin, esperamicin, shishijimicin and namenamicin, more preferably calicheamicin; or -D is auristatin, preferably MMAD, MMAE, MMAF or PF-06380101, more preferably MMAE or PF-06380101; or -D is a camptothecin, preferably selected from topotecan, silatecan, cositecan, exitecan, exitecan-S, DXd, SN-38, lortotecan, gimatecan, belotecan, rubitecan, AMDCPT and G-AMDCPT, more preferably exitecan or DXd, most preferably exitecan, More preferably, D is calicheamicin or exotecan, and more preferably, D is exotecan.

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

4. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody comprises: a V sequence having at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID No. 2, 6, 9, 11, 14, 18, 21, 24, 32 and 36; L A domain, and a V domain having at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID No. 1, 5, 10, 12, 13, 17, 23, 30 and 34. H domains, preferably wherein the antibody has complementarity determining regions (CDRs) that have at least 90% sequence identity.

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

6. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is 12C6 and / or comprises a light chain sequence according to SEQ ID No. 20 and a heavy chain sequence according to SEQ ID No. 19, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

7. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is 12C6a and / or comprises a light chain sequence according to SEQ ID No. 22 and a heavy chain sequence according to SEQ ID No. 19, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

8. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is 4D5 and / or comprises a light chain sequence according to SEQ ID No. 16 and a heavy chain sequence according to SEQ ID No. 15, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

9. The antibody conjugate according to any one of claims 1 to 2, wherein the antibody is 7C8 and / or comprises a light chain sequence according to SEQ ID No. 25 and a heavy chain sequence according to SEQ ID No. 26, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%.

10. The antibody conjugate according to any one of claims 1 to 9, wherein the linker L has the structure -(L 1 ) n -(L 2 ) o -(L 3 ) p -(L 4 ) q – in: -L 1 、L 2 、L 3 and L 4 each individually a linker, which together connect Z to D; - n, o, p and q are each independently 0 or 1, provided that n+o+p+q=1, 2, 3 or 4, Preferably wherein n=o=p=1; And among them: (a) Connector L 1 It is represented by: –(W) k –(A) d –(B) e –(A) f –(B) g –(C(O)) g –, or –(W) k –(A) d –(B) e –(A) f –(C(O)) g –N*[–(A) d’ –(B) e’ –(A) f’ –(C(O)) g” –]2, in: -d and d' are individually 0 or 1; -e and e' are independently integers ranging from 1 to 10; - f and f' are individually 0 or 1; - g and g' are independently integers ranging from 0 to 10; - k = 0 or 1, provided that if k = 1, then d = 0; -A is a sulfonamide group according to structure (23), Where a = 0 or 1, and R 13 Selected from hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 The group consisting of (hetero)arylalkyl groups, the C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl groups, C3–C 24 Alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally replaced by one or more selected from O, S and NR 14 The heteroatom spacing, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is D linked to N via a spacer moiety, preferably wherein said spacer moiety is -(B) g –(C(O)) g –(L 2 ) o –(L 3 ) p –(L 4 ) q – -W is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–, –C(O)(CH2) m C(O)–, –C(O)(CH2) m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, wherein m is an integer ranging from 0 to 10; -B is a –CH2–CH2–O– or –O–CH2–CH2– moiety, or (B) e is –(CH2–CH2–O) e1 -CH2-CH2- moiety, wherein e1 is an integer ranging from 1 to 10; -N* is a branched nitrogen atom, –(A) d –(B) e –(A) f –(C(O)) g’ Both instances of – are connected to it and both (C(O)) g’ Part of it is related to –(L 2 ) o –(L 3 ) p –(L 4 ) q –D connection, where L 2 、L 3 、L 4 , o, p, q and D are each selected individually; and / or (b) Connector L 2 is a peptide spacer, preferably comprising 1 to 5 amino acids, more preferably a dipeptide, tripeptide or tetrapeptide spacer, most preferably wherein L 2 It is represented by the general structure (L3): where R 17 =CH3 or CH2CH2CH2NHC(O)NH2; and / or (c) Connector L 3 is a self-immolative spacer, preferably a p-aminobenzyloxycarbonyl (PABC) derivative according to structure (L4), where R 21 It is H, R 26 or C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl groups, C3–C 10 Alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally replaced by one or more selected from O, S and NR 28 The heteroatom interval, where R 28 independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, Preferably, R 21 is H or C(O)R 26 , where R 26 =4-methyl-piperazine or morpholine, most preferably wherein R 21 It is H; and / or (d) Connector L 4 According to the structure –NR 22 –(C z -alkylene)-C(O)-aminoalkanoic acid spacer, wherein x is an integer in the range of 1 to 20 and R 22 is H or C1–C4 alkyl; or Connector L 4 According to the structure –NR 22 –(CH2–CH2–O) e6 –(CH2) e7 -C(O)-, wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R 22 is H or C1–C4 alkyl; or Connector L 4 According to the structure –NR 22 –(C z -alkylene)–NR 22 –(C(O)) h - a diamine spacer, wherein h is 0 or 1, x is an integer in the range of 1 to 10 and R 22 is H or C1–C4 alkyl.

11. The antibody conjugate according to any one of claims 1 to 10, wherein b=1 and conjugation is via the glycan of the antibody, preferably wherein j=0.

12. The antibody conjugate according to any one of claims 1 to 11, wherein Z-L-D has a structure selected from the group consisting of (X) to (XIII): The wavy line indicates the connection to Z, and L 2 、L 4 , o and q as defined in claim 10, Preferably wherein Z-LD has a structure selected from the group consisting of (Xa), (XIb), (XIIg), (XIIIe) and (XIIh): The wavy line indicates the connection to Z, and R 22 As defined in claim 10.

13. The antibody conjugate according to claim 12, which is according to structure (XII) or (XIII), and wherein o=1 and the antibody is labetuzumab as defined in claim 5, preferably wherein q=0.

14. The antibody conjugate according to claim 13, which is according to structure (XII) or (XIII), wherein q=0, o=1, L 2 is a peptide spacer containing 1 to 5 amino acids, the antibody is labetuzumab as defined in claim 5, and the payload is exitecan.

15. The antibody conjugate of claim 1, according to structure (XIIIe) as defined in claim 12, and wherein the antibody is cofetizumab as defined in claim 5, and the payload is exetotecan.

16. A method for preparing the antibody conjugate according to any one of claims 1 to 15, comprising: (i) an antibody comprising a γ-core N-acetylglucosamine (GlcNAc) portion and a compound of formula S(F) x -P compound in the presence of a catalyst, wherein y = 1, 2, 3 or 4, wherein S (F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q, x is 1 or 2 and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The α-GlcNAc moiety is transferred to the core-GlcNAc moiety to obtain a modified antibody of formula (26): AB–[GlcNAc(Fuc) w –S{F} x ] y (26) in -AB is an antibody that can target tumors expressing PTK7; -Fuc is fucose; -w is 0 or 1; and (ii) reacting the modified antibody with a compound according to structure (2): Q–L–D (2) in: -Q is the reactive moiety; -L is the linker connecting Z to D; -D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasters, KSP inhibitors, TLR agonists, dihydroindole and benzodiazepines Pyrrolobenzodiazepine The group consisting of dimers (PBD) and their analogs or prodrugs; to obtain the antibody conjugate according to structure (1).

17. The method according to claim 16, wherein the reaction of step (ii) is a nucleophilic reaction or a cycloaddition, preferably a 1,3-dipolar cycloaddition, and preferably wherein Q comprises or is an alkyne moiety and F comprises or is an azide moiety.

18. The method according to claim 16 or 17, wherein Q is a click probe comprising a (hetero)cycloalkyne moiety or a (hetero)cycloalkene moiety, preferably wherein the click probe Q is selected from (Q21) to (Q38) and (Q44) to (Q56): in -B is an anion; and - In (Q50) and (Q51), the R group on Si is an alkyl group or an aryl group; And wherein F is a click probe selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxythiophene and Sydney ketone, preferably F is an azide moiety.

19. A method for targeting cells expressing PTK7, comprising contacting the antibody conjugate according to any one of claims 1 to 15 with cells that may express PTK7, preferably wherein the cells are tumor cells expressing PTK7.

20. The method of claim 19, wherein said targeting of cells expressing PTK7 comprises one or more of the following: treating, imaging, diagnosing, preventing proliferation of, inhibiting, and reducing PTK7-expressing cells, particularly PTK7-expressing tumor cells.

21. The method of claim 19 or 20, wherein the subject has colon cancer, lung cancer, breast cancer, ovarian cancer, or esophageal cancer.

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