Antibody conjugates for targeting PTK7-expressing tumors

Abstract

The present invention relates to an antibody conjugate that is particularly suitable for targeting PTK7 expressing cells, especially tumor cells. The antibody conjugate according to the present invention has the structure (1): AB-[(L6) b -{ZLD} x ] y (1) where AB is an antibody capable of targeting PTK7-expressing tumors, L is a linker connecting Z to D, Z is a connecting group, and L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-; D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulin, BCL-XL inhibitors, hemiasterlin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; b is 0 or 1; x is 1 or 2; and y is 1, 2, 3, or 4. The present invention further relates to methods for preparing the antibody conjugates of structure (1) and applications of the antibody conjugates of structure (1).

Description

[Technical field]

[0001] The present invention is in the field of bioconjugation. More particularly, the present invention relates to antibody-drug conjugates for targeted treatment of patients with cancer, particularly PTK7-expressing tumors. [Background technology]

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

[0003] Current ADCs are commonly prepared by a variety of conjugation techniques (summarized in Figure 1), primarily based on conjugation to cysteine ​​side chains bearing maleimides or lysine side chains bearing activated esters. To generate ADCs based on native cysteines that naturally participate in disulfide bonds, the thiols in the side chains can be liberated by exposure of the antibody to a suitable reducing agent, such as TCEP or DTT, followed by treatment with a maleimide-functionalized linker drug. The resulting ADCs typically consist of a mixture of positional isomers if only the total of the eight free thiols are not comprehensively alkylated. Alternatively, to generate site-specific ADCs, antibodies can be generated by mutating one or more amino acids to cysteines at defined positions in the sequence, whose side chains can be selectively liberated for alkylation by a reduction-oxidation sequence. Commonly used cysteines for site-specific conjugation are LC-41C (light chain 41C), HC-41C (heavy chain 41C), LC-80C, HC-118C, HC-265C, HC-140C, LC-149C, LC-124C, LC-180C, HC-190C, HC-160C, LC-183C, HC-290C, LC-205C, HC-220C, HC-239C, HC-442C. Additional cysteines, such as HC-i239C, can also be inserted into the sequence. In addition to maleimides as alkylating agents, reactions of cysteine ​​side chains with haloacetamides or vinylbenzene derivatives have also been reported. In addition to reacting with natural amino acid side chains, certain unnatural (non-standard) amino acids can also be engineered into the amino acid sequence of an antibody, thereby providing unique handles for chemical conjugation such as ketones, acetylenes, azides, cyclic alkynes or cyclic alkenes for reaction with oximes, azides, alkynes or tetrazines, respectively. However, the drawback of the latter approach is that the native sequence of the antibody must be redesigned, which, in addition to being time-consuming and expensive, can lead to instability problems.

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

[0005] Glycan-mediated chemoenzymatic conjugation, including initial trimming of the glycan, is known in the art and described by van Geel et al, Bioconjugate Chem. 2015, 26, 2233, and is shown diagrammatically in FIG. 2. Briefly, monoclonal antibodies are treated with endoglycosidases to trim the glycan to the core GlcNAc (directly attached to Asn-297), followed by the transfer of the azido-modified sugar under the action of a glycosyltransferase. Various structures of UDP-azido sugars are shown in FIG. 3. One particularly suitable combination involves the transfer of GalNAz 2b (2-azidoacetyl-N-galactosamine) under the action of the mutant galactosyltransferase GalT (Y289L) disclosed in WO2007 / 095506, EP2911699B1, and van Geel et al. Another useful combination involves GlcNAz with α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT1) and α-1,6-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase (MGAT2), as disclosed in WO2018 / 126092 and WO2021 / 248048.

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

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

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

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

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

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

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

[0013] Antibodies against PTK7 are known in the art, such as those disclosed in US9777070B2 (H23 and H24, also known as cofetuzumab), 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 (cofetuzumab peridotin), based on a humanized anti-PTK7 antibody and an auristatin microtubule inhibitor payload (Aur0101). PF-06647020 has been shown to induce long-lasting tumor regression in patient-derived tumor xenograft preclinical models, and to induce an acceptable safety profile and preliminary clinical activity in previously treated patients with locally advanced / metastatic, PTK7-positive NSCLC, TNBC, and platinum-resistant OvCa, administered every 2 or 3 weeks. Summary of the Invention [Problem to be solved by the invention]

[0015] The present inventors have developed an antibody conjugate that is highly suitable for targeting PTK7 expressing cells, especially tumors, and is therefore highly suitable for the treatment of PTK7 positive cancers, especially colon cancer, lung cancer, breast cancer, ovarian cancer and esophageal cancer. [Means for solving the problem]

[0016] In a first aspect, the present invention relates to an antibody conjugate. In this regard, in a second aspect, the present invention relates to a process for preparing an antibody conjugate according to the present invention. In a third aspect, the present invention relates to a method for targeting PTK7-expressing cells. In this regard, the first medical use of the antibody conjugate according to the present invention, as well as the second medical use for the treatment of cancer. In a last aspect, the present invention relates to the use of the conjugation mode to increase the therapeutic index of the antibody conjugate in the treatment of PTK7-expressing tumors.

[0017] definition As used in this description and claims, the verb "comprise" and its conjugations are used in its open-ended sense, meaning that items following the word are included but not excluding items not specifically mentioned.

[0018] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one of the element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0019] A linker is defined herein as a moiety that connects (covalently bonds) two or more elements of a compound. A linker may include one or more spacer moieties. A spacer moiety is defined herein as a moiety that separates (i.e., provides distance between) two (or more) parts of a linker and covalently bonds them to each other. A linker can be, for example, part of a linker construct, a linker conjugate, a linker payload (e.g., a linker drug) or an antibody conjugate, as defined below.

[0020] "Hydrophilic group" or "polar linker" is defined herein as any molecular structure that contains one or more polar functional groups that impart improved polarity and therefore improved water solubility to the molecule to which it is attached. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonic acid groups, phosphate groups, acylsulfamide groups or carbamoylsulfamide groups. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency and improved pharmacokinetics that result in less aggregation, higher efficacy and in vivo tolerability when incorporated into antibody-drug conjugates.

[0021] The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced with a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharma- ceutically acceptable salt, although this is not necessary for salts that are not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation with the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term "pharma-ceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts may be derived from pharma-ceutically acceptable inorganic or organic bases and pharma-ceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharma- ceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0022] The term "enediyne" or "enediyne antibiotic" or "enediyne-containing cytotoxin" refers to any cytotoxin characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule known in the art, including neocarzinostatin (NCS), C-1027, kedarcidin (KED), madulopeptin (MDP), N1999A2, sporolide (SPO), cyanosporacids (CYA and CYN), as well as physiolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamemycin, shishijimicin, and unicaramycin (UCM).

[0023] The term "alkylaminosugar," as used herein, refers to a tetrahydropyranyl moiety connected through its 2-position to an alcohol functional group, thereby forming an acetal functional group, and further substituted with (at least) one N-alkylamino group at positions 3, 4 or 5. An "N-alkylamino group" in this context refers to an amino group bearing one methyl, ethyl, or 2-propyl group.

[0024] The term "click probe" refers to a functional moiety capable of undergoing a click reaction, i.e., two compatible click probes click with each other such that they are covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of the present invention, a click probe Q in a compound according to the invention can react with a click probe F on a (modified) protein such that, upon the occurrence of a click reaction, a conjugate is formed in which the protein is conjugated to the compound according to the invention. Here, F and Q are compatible click probes.

[0025] An "acylsulfamide moiety" is defined herein as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamoylated at one end of the molecule and N-alkylated (mono- or bis) at the other end of the molecule. In the context of the present invention, particularly in the examples, this group is also referred to as "HS."

[0026] A "coding sequence," or a sequence that "encodes" an expression product, e.g., an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, upon expression, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0027] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as, for example, promoter sequences, which determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may cover genomic sequences that code for proteins, i.e., sequences that include regulatory elements, promoters, introns, and exon sequences.

[0028] The term "glycoprotein" is used herein in its ordinary scientific sense to refer to a protein that contains one or more mono- or oligosaccharide chains ("glycans") covalently attached to the protein. The glycans may be attached to a hydroxyl group on the protein (O-linked glycans), e.g., the hydroxyl group of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline, or to an amide functional group on the protein (N-glycoproteins), e.g., asparagine or arginine, or to a carbon on the protein (C-glycoproteins), e.g., tryptophan. A glycoprotein may contain more than one glycan, may contain a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may contain a combination of N-linked glycans, O-linked glycans, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and thus qualify as glycoproteins. Examples of glycoproteins include PSMA (prostate specific membrane antigen), CAL (Candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins and antibodies.

[0029] The term "glycan" is used herein in its normal scientific sense to refer to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term glycan refers to the carbohydrate moiety of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of one sugar, which may be without further substitution (monosaccharide) or may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when a longer glycan chain is linked to a protein, the glycan is also considered herein as a glycan. The glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar that is directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but is attached to a single other sugar, i.e., has no further sugar substituents at one or more of the other hydroxyl groups, is called a terminal sugar. For the avoidance of doubt, there may be multiple terminal sugars in the oligosaccharide of a glycoprotein, but only one core sugar. A glycan may be an O-linked glycan, an N-linked glycan, or a C-linked glycan. In an O-linked glycan, the monosaccharide or oligosaccharide glycan is attached to an O atom in an amino acid of the protein, typically through a hydroxyl group of serine (Ser) or threonine (Thr). In an N-linked glycan, the monosaccharide or oligosaccharide glycan is attached to a protein through an N atom in an amino acid of the protein, typically through the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In a C-linked glycan, the monosaccharide or oligosaccharide glycan is attached to a C atom in an amino acid of the protein, typically the C atom of tryptophan (Trp).

[0030] The term "antibody" (AB) is used herein in its normal scientific sense. An antibody is a protein produced by the immune system that is capable of recognizing and binding to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" includes whole antibodies, but is also intended to include antibody fragments, such as truncated antibodies, scFv-Fc fragments, minibodies, diabodies, or antibody Fab fragments, F(ab')2, Fv fragments, or Fc fragments derived from scFv. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0031] An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). Light chains contain two domains or regions: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to antigens. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g., human G1m1, G1m2, Gm3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3). Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2 or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. Antibodies can be engineered in the Fc domain to enhance or abolish binding to Fc-gamma receptors, as summarized by Saunders et al. Front. Immunol. 2019, 10, doi:10.3389 / fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131-141.For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. Elimination of binding to Fcγ receptors can also be achieved by mutating the N297 amino acid to any other amino acid except asparagine, mutating the T299 amino acid to any other amino acid except threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies, for example, with PNGase F or endoglycosidase. Immunoglobulins can be from any species, including from human, mouse, or rabbit. Each chain contains a different sequence domain.

[0032] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where portions of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the entire length of the reference sequence.

[0033] The term "CDR" refers to complementarity determining region, and the specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. An antibody binding site is composed of residues that are primarily from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) also influence the overall domain structure and thus the binding site. Thus, complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin have three CDRs, CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0034] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody molecule of a single amino acid sequence directed against a particular antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of a B cell or hybridoma, but may also be recombinant, i.e., produced by protein engineering.

[0035] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises the VH and VL domains of an antibody from a non-human animal associated with the CH and CL domains of another antibody, in one embodiment a human antibody. As a non-human animal, any animal can be used, such as mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also represent multispecific antibodies, having specificity for at least two different antigens.

[0036] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified to avoid or minimize immune responses in humans, for example by replacing certain amino acids in the framework regions of the VH and VL domains. The constant domains of a humanized antibody are most often human CH and CL domains. A "fragment" of a (conventional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0037] 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, with no observable kinase activity across species. [Brief description of the drawings]

[0038] [Figure 1] Shown below is a representative set of reactive groups (F) that, when present in a biomolecule, yield a connecting group Z (1a-1h) upon reaction with reactive group Q. The functional group F can be artificially introduced (engineered) into the biomolecule at any position of interest. [Diagram 2] It is shown diagrammatically how an antibody conjugate can be obtained from any monoclonal antibody in a two-step process. In the first step, an azide-modified UPD-Gal or UDP-GalNAc can be conjugated to the monoclonal antibody in a one-pot process involving (a) trimming of the glycan (to the core GlcNAc) by an endoglycosidase and (b) attachment of the azido sugar under the action of a glycosyltransferase (galactosyltransferase or a mutant thereof or GalNAc transferase), thereby generating a β-glycosidic 1-4 linkage between the azide-modified GalNAc and the GlcNAc. In the second step, the azide-modified antibody can be reacted with an appropriately functionalized cyclooctyne, thereby generating the antibody conjugate. [Diagram 3] For example, structures of some derivatives of the UDP-sugar of galactosamine are shown that can be modified with a 3-mercaptopropionyl group (2a), an azidoacetyl group (2b), or an azidodifluoroacetyl group (2c) at the 2-position of N-acetylgalactosamine, or with an azido group (2d) at the 6-position. [Figure 4] Preferred choices for reactive moiety Q are cyclooctynes ​​(A-T) suitable for metal-free click chemistry. [Diagram 5]5 shows a set of exemplary toxic payloads for conjugation to various PTK7-targeting monoclonal antibodies according to the present invention. The attachment point of the linker (to the amino group present in the payload) is indicated by an arrow. A preferred conjugate according to the present invention contains a payload that includes an attachment point as shown in FIG. 5. [Figure 6] 1 shows the structures of various camptothecins, which are preferred payloads in the context of the present invention. [Figure 7] 1 shows structures of BCN-linker drugs used to prepare ADCs via click chemistry to azido-sugar remodeled antibodies with the following payloads (3=exatecan, 4=MMAE, 5a=calicheamicin γ1 I, 5b=glycine-calicheamicin γ1 I, 6=6-aminohexanoyl-maytansinoid). [Figure 8A] Background corrected binding of cofetuzumab to hPTK7 and cPTK7 is shown. [Figure 8B] 1 shows comparable binding to hPTK7 for all cofetuzumab-based ADCs compared to the corresponding mAbs. [Figure 9] Figure 1 shows in vitro potency data for A431 cells treated with increasing concentrations of the ADCs cofetuzumab-4, cofetuzumab-5b, and negative control B12-3. Both cofetuzumab ADCs show cell killing with IC50 values ​​of 3.9 nM and 0.1 nM, respectively. [Figure 10A] In vivo efficacy data of the PTK7 expressing NCI-H446 tumor model over time (Figure 10A: up to t=14 days, Figure 10C: up to t=44 days). Cofetuzumab-3 results in tumor regression with a clear dose response (2.59, 5.18, and 10.36 mg / kg). Administration of cofetuzumab-5b ADC also shows a dose response (2.59 and 5.18 mg / kg) that results in tumor growth delay. Other ADCs, including cofetuzumab-Aur0101, show only marginal tumor growth delay (similar to isotype control ADC B12-3). [Figure 10B] Mouse body weight over time is shown (FIG. 10B: up to t=14 days, FIG. 10D: up to t=28 or 44 days). [Figure 10C] In vivo efficacy data of the PTK7 expressing NCI-H446 tumor model over time (Figure 10A: up to t=14 days, Figure 10C: up to t=44 days). Cofetuzumab-3 results in tumor regression with a clear dose response (2.59, 5.18, and 10.36 mg / kg). Administration of cofetuzumab-5b ADC also shows a dose response (2.59 and 5.18 mg / kg) that results in tumor growth delay. Other ADCs, including cofetuzumab-Aur0101, show only marginal tumor growth delay (similar to isotype control ADC B12-3). [Figure 10D] Mouse body weight over time is shown (FIG. 10B: up to t=14 days, FIG. 10D: up to t=28 or 44 days). [Figure 11] Binding of 4D5-3, 12C6a-3, 12C6-3 and 7C8-3 to hPTK7, cPTK7 and rPTK7 is shown. 12C6-3 shows the highest binding affinity to both human and cynomolgus PTK7 in comparison. 12C6a-3 shows no binding at all. 4D5-3 and 7C8-3 show similar binding to hPTK7 and cPTK7, while 7C8 clearly has a higher binding affinity to rPTK7. [Figure 12A] Figure 1 shows in vivo efficacy data for the PTK7 expressing NCI-H446 tumor model over time. 12C6-3 is similar to vehicle and shows no tumor growth inhibition. Both 7C8-3 and 4D5-3 show limited tumor growth delay. Cofetuzumab-3 shows tumor growth inhibition but also shows early tumor regrowth. 12C6-3 shows 99.7% tumor growth inhibition by day 40. [Figure 12B] Mouse body weight over time is shown. [Figure 12C] 1 shows survival over time in a Kaplan-Meier plot for the NCI-H446 model. [Figure 13A]Figure 1 shows in vivo efficacy data of PTK7 expressing BR1282 PDX tumor model over time. 12C6-3 has better tumor growth inhibition rate than all 7C8-3, 4D5-3 and 12C6a-3, and also compared to cofetuzumab-Aur0101. Compared to cofetuzumab-3, both ADCs show similar response and show similarly good tumor growth inhibition. [Figure 13B] Mouse body weight over time is shown. [Figure 14A] Shows in vivo efficacy data for PTK7 expressing NCI-H446 tumor model over time.Olaparib as single treatment shows no significant effect.Both 12C6-3 and 12C6-9 show tumor growth inhibition / stagnation.However, the combination of Olaparib and 12C6-3 shows a clear additive effect, resulting in complete response in 6 out of 8 mice at day 21, and the study is still ongoing. [Figure 14B] Mouse body weight over time is shown. [Figure 15] Figure 1 shows the in vivo tolerability data of 12C6-3 in female Sprague Dawley rats over time. No toxicity or weight loss was observed at any concentration. Thus, the maximum tolerated dose is greater than 150 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] In a first aspect, the present invention relates to an antibody-conjugate of general structure (1): AB-[(L 6 ) b -{ZLD} x ] y (1) During the ceremony, -AB is an antibody capable of targeting PTK7-expressing tumors, - L is a linker connecting Z to D, -Z is a connecting group; -L 6 -GlcNAc(Fuc) w -(G) j -S-(L7 ) w’ -, in which G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-, D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; -b is 0 or 1, -x is 1 or 2, -y is 1, 2, 3, or 4.

[0040] Salts, preferably pharma-ceutically acceptable salts, of the antibody conjugates according to structure (1) are also contemplated in the present invention.

[0041] In a second aspect, the present invention relates to a process for preparing an antibody conjugate according to the present invention, comprising reacting a compound according to general structure (2) with an antibody (3). The compound according to general structure (2) comprises a reactive moiety Q and an antibody-reactive moiety F capable of reacting with Q in a conjugation reaction, where Q and F react to form a linking group Z. In this reaction, a conjugate according to general structure (1) is formed. The process according to this aspect relates to the following bioconjugation reaction: AB-[(L 6 ) b -{F} x ] y +ZLD→AB-[(L 6 ) b -{ZLD} x ] y (3) (2) (1)

[0042] In the following, an antibody conjugate according to structure (1) is first defined. The structural features of an antibody conjugate according to structure (1) also apply to a compound according to structure (2) and an antibody according to structure (3), since they are invariant in the conjugation reaction, except for the reactive moieties F and Q, which are converted to a linking group Z upon reaction of a compound according to structure (2) with an antibody according to structure (3).

[0043] In a third aspect, the present invention relates to an application antibody conjugate according to structure (1) for targeting PTK7 expressing cells. In this regard, the present invention relates to a first medical use and a second medical use of an antibody conjugate according to structure (1).

[0044] As will be appreciated by one of skill in the art, the definitions of chemical moieties, and their preferred embodiments, apply to all aspects of the present invention.

[0045] Antibody conjugates of general structure (1) In a first aspect, the present invention relates to an antibody-conjugate of general structure (1): AB-[(L 6 ) b -{ZLD} x ] y (1) During the ceremony, -AB is an antibody capable of targeting PTK7-expressing tumors, -b is 0 or 1, -L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-, -Z is a connecting group; - L is a linker connecting Z to D, D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; -x is 1 or 2, -y is 1, 2, 3, or 4.

[0046] Antibody AB The antibody conjugate according to the present invention contains an antibody capable of targeting PTK7 expressing cells, particularly tumor cells. PTK7 is a known target for cancer therapy. The term "express" is commonly used in the art and refers to overexpression of a target relative to expression in healthy tissue. An antibody capable of targeting PTK7 expressing tumors is also referred to as an anti-PTK7 antibody, a PTK7 targeting antibody, or a PTK7 binding antibody. An anti-PTK7 antibody selectively binds to PTK7 expressing cells. Anti-PTK7 antibodies are known in the art and any suitable one can be used in the context of the present invention.

[0047] The Fc regions of these antibodies may have one or more mutations, such as 0-10 mutations or 0-5 mutations. Mutations that alter binding to the FcRn receptor to modulate the half-life of the antibody are particularly preferred. For example, inclusion of Met to Tyr, Ser to Thr, and Thr to Glu mutations in the region of amino acids 254-260 of the heavy chain, often referred to as YTE, in an IgG1 Fc increases the binding of the antibody to human FcRn by approximately 11-fold, thereby increasing the circulating half-life by approximately 3.5-fold. For example, the YTE mutations in cofetuzumab are Met254Tyr, Ser256Thr, and Thr258Glu. Thus, in one embodiment, the antibody has a circulating half-life of 2.5×10 -6 Less than M, preferably 0.05 to 0.99 × 10-6 M, more preferably 0.1 to 0.49 × 10 -6 M, most preferably 0.2 to 0.4 × 10 -6 Apparent human FcRn binding affinity K in the range of M D,app The apparent binding affinity K D,app can be determined according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, the antibody AB is a YTE variant of a preferred antibody as defined above or below.

[0048] In a preferred embodiment, the antibody is cofetuzumab. Cofetuzumab may 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, with a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0049] In a preferred embodiment, the antibody is 12C6. 12C6 may 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, with sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0050] In a preferred embodiment, the antibody is 12C6a. 12C6a may 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, with a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0051] In a preferred embodiment, the antibody is 4D5. 4D5 may 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, with sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0052] In a preferred embodiment, the antibody is 7C8. 7C8 may 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, with sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0053] In a preferred embodiment, the antibody is Hu23. Hu23 may 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, with sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0054] In a preferred embodiment, the antibody is Hu58. Hu58 may 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, with sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably with exatecan as payload D.

[0055] Thus, in a preferred embodiment, the antibody is selected from cofetuzumab, 12C6, 12C6a, 4D5, 7C8, Hu23, and Hu58, all as defined above, more preferably from cofetuzumab, 12C6, 4D5, and 7C8, even more preferably from cofetuzumab and 12C6. In one embodiment, the antibody is cofetuzumab. In one embodiment, the antibody is 12C6.

[0056] Preferred antibodies have their V L Domain and V HIn a preferred embodiment, the antibody is defined by a variable domain, which together form an antigen-binding variable domain. Thus, in a preferred embodiment, the antibody is a variable domain selected from the group consisting of SEQ ID NOs: 2, 6, 9, 11, 14, 18, 21, 24, 32, and 36. L domain and a V selected from the group consisting of SEQ ID NOs: 1, 5, 10, 12, 13, 17, 23, 30, and 34. H In a particularly preferred embodiment, the antibody comprises the V domain of SEQ ID NO:6, which has a sequence identity of at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, and most preferably at least 99% or 100%. L Domain and V of SEQ ID NO:5 H domains, the sequence identity being at least 70%, preferably at least 75%, or at least 80%, more preferably at least 85% or at least 90% or at least 95%, and most preferably at least 99% or even 100%.

[0057] The sequence identity described above is L Domain and V H The term refers to the complete sequence of the domain. The entire sequence of these domains allows for some variation in the sequence without compromising binding to PTK7, however, it is preferred that the complementarity determining regions (CDRs) have a higher sequence identity to ensure that binding to PTK7 is not significantly compromised. The positions of the CDRs are shown in the table below. Thus, the antibody can be selected from the group consisting of SEQ ID NOs: 2, 6, 9, 11, 14, 18, 21, 24, 32, and 36. L domain and a V selected from the group consisting of SEQ ID NOs: 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 Preferably, the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.L Domain and V H It will be appreciated that the domains may be combined with appropriate constant domains to form a complete antibody.

number

number

[0058] In a particularly preferred embodiment, the antibody comprises the V 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:10 H 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 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 In the present specification, the sequence identities defined above for the complete sequence and for the CDRs apply.

[0059] Alternatively, an antibody is defined by its light and heavy chains, which together form the antibody. Thus, in a preferred embodiment, the antibody comprises a light chain selected from the group consisting of SEQ ID NO: 4, 8, 16, 20, 22, 26, 33, and 37, and a heavy chain selected from the group consisting of SEQ ID NO: 3, 7, 15, 19, 25, 31, and 35, with a sequence identity of at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a further preferred embodiment, the antibody comprises a light chain of SEQ ID NO: 8 and a heavy chain of SEQ ID NO: 7, with a sequence identity of at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.

[0060] The above sequence identity refers to the complete sequence of the light and heavy chains. The entire sequence of these chains allows some variation in the sequence without compromising binding to PTK7, but it is preferred that the CDRs have a higher sequence identity to ensure that binding to PTK7 is not significantly compromised. The positions of the CDRs are shown in the table below. Thus, the antibody preferably comprises a light chain selected from the group consisting of SEQ ID NO: 4, 8, 16, 20, 22, 26, 33, and 37, and a heavy chain selected from the group consisting of SEQ ID NO: 3, 7, 15, 19, 25, 31, and 35, and the sequence identity of the CDRs is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%.

number

number

[0061] In a particularly preferred embodiment, 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 identities defined above for the complete sequences and for the CDRs apply.

[0062] Linker L 6 When the reactive group F is directly attached to the antibody, or even to a part of the antibody structure, a linker L connecting AB to F (in the case of an antibody of structure (3)) or AB to Z (in the case of a conjugate of structure (1)) can be used. 6 is absent and b=0. This is the case, for example, for cysteine ​​and lysine conjugations. Alternatively, the linker L connecting AB to F (in the case of an antibody of structure (3)) or AB to Z (in the case of a conjugate of structure (1)) 6 A reactive group F can also be introduced into the antibody using 6 exists and b=1. L 6 If present, the reactive group F is typically introduced into the glycan of the antibody. This is the case, for example, for conjugation via an artificially introduced reactive group F, such as, for example, using transglutaminase or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase). For example, the modified sugar residue S(F) x may be introduced into the glycan to extend the glycan with one monosaccharide residue S that introduces x reactive groups F onto the glycan of the antibody. In the most preferred embodiment, conjugation occurs via the glycan of the antibody, and b=1. The site of conjugation is preferably on the heavy chain of the antibody.

[0063] If present, L 6is a linker connecting AB to F or Z, -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. Typically, L 6 is formed at least in part by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at the asparagine residue at or near position 297 of the heavy chain, which is modified by complex-type glycans. This naturally occurring glycosylation site of the antibody is preferably used, although other glycosylation sites, including artificially introduced ones, may also be used in the linker L. 6 Therefore, in a preferred embodiment, L 6 is attached to an amino acid of the antibody located at a position within 250 to 350 of the heavy chain, preferably within 280 to 310 of the heavy chain, more preferably within 295 to 300 of the heavy chain, and most preferably at position 297 of the heavy chain.

[0064] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan of an antibody, or a part thereof. Thus, the -GlcNAc(Fuc) of the glycan w -(G) j- is typically derived from the original antibody, GlcNAc is an N-acetylglucosamine moiety, and Fuc is a fucose moiety. Fuc is typically attached to GlcNAc via an α-1,6-glycosidic bond. Usually, the antibody can be fucosylated (w=1) or non-fucosylated (w=0). In the context of the present invention, the presence of the fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue, which may also be referred to as the core-GlcNAc residue, is the monosaccharide that is directly attached to the peptide portion of the antibody.

[0065] S is core-GlcNAc(Fuc) w The moiety may be directly attached to the core-GlcNAc(Fuc) moiety, i.e., j=0, which means that the remainder of the glycan is connected to the core-GlcNAc(Fuc) moiety before S is attached. w Such trimming of glycans is well known in the art and can be accomplished by the action of endoglycosidases. Alternatively, the core-GlcNAc(Fuc) w There is one or more monosaccharide residues between the moiety and S, i.e. j is an integer in the range of 1 to 10, preferably j=2 to 5. In a preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer ranging from 2 to 5. j is typically linked to GlcNAc(Fuc) via a β-1,4 bond. wIn a preferred embodiment, j is 3, 4 or 5. Any monosaccharide that may be present in a glycan may be used as G, but each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and N-acetylneuraminic acid. More preferred choices for G are galactose, N-acetylglucosamine, and mannose. The inventors have found that antibody conjugates with j less than 4 exhibit no or little binding to Fc-gamma receptors, while antibody conjugates with j in the range of 4 to 10 bind to Fc-gamma receptors. Thus, by selecting a particular value of j, a desired degree of binding to Fc-gamma receptors can be obtained. Thus, it is preferred that j=0, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably j=0, 3, 4 or 5, most preferably the antibody is trimmed and j=0.

[0066] S is a sugar or sugar derivative. The term "sugar derivative" is used herein to indicate a monosaccharide sugar, i.e. a derivative of a monosaccharide sugar, including a substituent and / or a functional group. Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), also called N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Preferably, S is selected from Glc, Gal, GlcNAc, and GalNAc. In a particularly preferred embodiment, S is GalNAc.

[0067] x is an integer indicating the number of linking groups Z (of conjugate (1)) or reactive groups F (of antibody (3)) attached to the sugar (derivative) S. Thus, an antibody according to the invention contains a moiety S comprising x reactive moieties F. Each of these reactive moieties F reacts with a reactive moiety Q of a compound according to general structure (2) such that x linking groups Z are formed, and x compounds according to general structure (2) are attached to a single occurrence of S. x is 1 or 2, preferably x=1.

[0068] The linking group Z (in the case of conjugate (1)) or the reactive group F (in the case of antibody (3)) may be directly attached to S or may be linked by a linker L present between S and Z or F. 7 may be present. 7 is a linker connecting S to Z. 7 may be present (w'=1 or 2) or absent (w'=0). Typically, each moiety Z is linked to a linker L 7 , so that in one embodiment w′=0 x. 7 is absent and each binding site Z is directly attached to S. If present, L 7 teeth, It may 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.

[0069] y is an integer representing the number of sugar(s) (derivative(s)) S, each of which has x reactive groups F or is connected to x connecting groups Z, which are connected to the antibody. y is 1, 2, 3, or 4, preferably y=2 or 4, and most preferably y=2. Thus, the antibody comprises y moieties S, each of which comprises x reactive moieties F. Each of these reactive moieties F reacts with a reactive moiety Q of a compound according to general structure (2), resulting in the formation of x+y connecting groups Z, and x+y compounds according to general structure (2) being attached to a single antibody. Each compound according to general structure (2) can be, for example, a nitrogen atom N in L.* By branching of D, multiple payloads can be included. Each compound according to general structure (2) preferably contains one or two occurrences of D, most preferably two occurrences of D. In a particularly preferred embodiment, the linker L 1 The second occurrence of D is connected to a branched nitrogen atom, N * Contains:

[0070] The amount of payload (D) molecules bound to a single antibody is known in the art as DAR (drug-antibody ratio). In the context of the present invention, DAR is preferably an integer in the range of 1 to 8, more preferably 2 or 4, most preferably DAR=4. Alternatively, DAR is preferably an integer in the range of (x+y) to [(x+y)×2], most preferably DAR=[(x+y)×2]. For preferred values ​​of x of 1 and y of 2, DAR is preferably 4. It will be understood that these are theoretical DAR values, and in practice, the DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as stochastic mixtures of antibody-drug conjugates, DAR values ​​vary between individual conjugates, and depending on the conjugation technique used, the DAR may have a wide distribution (e.g., DAR=0-10) or a narrow distribution (e.g., DAR=3-4). For such mixtures, DAR often refers to the average DAR of the mixture. This is well known in the field of bioconjugation. However, if the conjugation occurs via a glycan (i.e., b=1 and L 6 is present), the antibody conjugate according to the invention has a DAR close to the theoretical DAR. For example, if the theoretical DAR is 4, DAR values ​​of greater than 3.6 or greater than 3.8 are readily obtained, indicating that most of the antibodies in the reaction mixture have reacted completely, resulting in a DAR of 4.

[0071] Connecting group Z Z is a connecting group that covalently connects both parts of the conjugate according to the invention. The term "connecting group" in this specification refers to a structural element resulting from the reaction between Q and F that connects one part of a conjugate with another part of the same conjugate. As will be understood by those skilled in the art, the nature of the connecting group depends on the type of reaction that results in the connection between the parts of the compound. As an example, if the carboxyl group of RC(O)-OH reacts with the amino group of H2N-R' to form RC(O)-N(H)-R', R is connected to R' through the connecting group Z, and Z can be represented by the group -C(O)-N(H)-. The connecting group Z can take any form since it is derived from the reaction between Q and F.

[0072] Since more than one reactive moiety F may be present or introduced in the antibody, the antibody conjugate according to the invention may contain more than one payload D, such as 1 to 8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D per biomolecule. The number of payloads is typically an even number, taking into account the symmetrical nature of antibodies. In other words, if one side of the antibody is functionalized with F, the symmetrical counterpart will also be functionalized. Alternatively, if the naturally occurring thiol group of a cysteine ​​residue of a protein is used as F, the value of m can be any and can vary between individual conjugates.

[0073] In the compound according to structure (1), the connecting group Z is optionally connected to L via a linker L. 6 D is linked to AB via AB. Numerous reactions for attaching reactive group Q to reactive group F are known in the art. As a result, a wide variety of connecting groups Z may be present in the conjugates according to the invention. In one embodiment, the reactive group Q is preferably selected from the options above, as shown in FIG. 1, and the complementary reactive group F and the connecting group Z thus obtained are known to those skilled in the art. When a linker conjugate containing Q is conjugated to a biomolecule containing a complementary reactive group F, F and Q, as well as the connecting group Z, present in the bioconjugate, 3Some examples of suitable combinations are shown in FIG.

[0074] For example, when F contains or is a thiol group, the complementary group Q includes N-maleimidyl and alkenyl groups, and the corresponding connecting group Z is as shown in Figure 1. When F contains or is a thiol group, the complementary group Q also includes an arenamide group.

[0075] For example, when F includes or is an amino group, the complementary group Q includes a ketone group and an activated ester group, and the corresponding connecting group Z is as shown in FIG.

[0076] For example, when F contains or is a ketone group, the complementary groups Q include (O-alkyl)hydroxylamino groups and hydrazine groups, and the corresponding connecting groups Z are as shown in FIG.

[0077] For example, when F contains or is an alkynyl group, the complementary group Q contains an azide group and the corresponding connecting group Z is as shown in FIG.

[0078] For example, when F comprises or is an azide group, the complementary group Q comprises an alkynyl group and the corresponding connecting group Z is as shown in FIG.

[0079] For example, when F comprises or is a cyclopropenyl group, a transcyclooctene group, or a cyclooctyne group, the complementary group Q comprises a tetrazinyl group and the corresponding connecting group Z is as shown in Figure 1. In these particular cases, Z is merely an intermediate structure that expels N2, thereby generating a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0080] Further suitable combinations of F and Q, and the nature of the resulting connecting group Z3, are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), which is incorporated by reference, in particular in Chapter 3, pages 229 to 258. A list of complementary reactive groups suitable for bioconjugation processes is disclosed in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), Chapter 3, pages 230 to 232, Table 3.1, the contents of which are expressly incorporated herein by reference.

[0081] In a preferred embodiment, the linking group Z is obtained by a cycloaddition or nucleophilic reaction, preferably the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition, or the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Such a cycloaddition or nucleophilic reaction occurs via a reactive group F connected to S and a reactive group Q connected to D via L. Conjugation reactions via cycloaddition or nucleophilic reactions are known to those skilled in the art, who will be able to select the appropriate reaction partners F and Q and will understand the nature of the resulting linking group Z.

[0082] In a first preferred embodiment, Z is formed by cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reaction) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art and the skilled person knows how to carry them out.

[0083] Preferably, Z comprises a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide or imide groups. It is particularly preferred that a triazole moiety is present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e. is formed from Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. is formed from Q comprising a (hetero)cycloalkene moiety. In a preferred embodiment, Z has the structure (Z1). [ka]

[0084] Here, the bonds shown as --- are single or double bonds. the ring Z is obtained by cycloaddition, preferably the ring Z is selected from (Za) to (Zj) as defined below, ** The carbon atoms labeled with correspond to the two carbon atoms of the bond shown as --- of (Z1) to which the ring Z is fused, -R15 are independently hydrogen, halogen, -OR 16 , NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by (-) is an anion, and each R 31 are individually, R 15 or a connection to D connected via L, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; -v=an integer in the range of 8 to 16, Ring A is formed by cycloaddition and is preferably selected from (Za)-(Zj).

[0085] When the bond shown as --- is a double bond, it is preferred that u+u'=4, 5, 6, 7, or 8. * The wavy bond labeled with is connected to S, ** The wavy bond labeled with is connected to L.

[0086] It is particularly preferred that Z comprises a (hetero)cycloalkene moiety, i.e., the bond shown as --- is a double bond. In a preferred embodiment, Z is selected from structures (Z2) to (Z20) shown below: [ka]

[0087] Here, the connection to L is shown as a wavy coupling. (-) is an anion, preferably a pharma- ceutically acceptable anion. Ring Z is formed by a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za) to (Zm) shown below, where: ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane ring (Z2) to (Z20) to which the ring Z is fused. Preferred rings Z are selected from (Za) to (Zj), more preferably (Za), (Zd) and (Zh), and most preferably ring Z has the structure (Za). The connecting group Z is formed in the context of this embodiment by reaction with a (hetero)cycloalkyne, and therefore the bond shown above as --- is a double bond. [ka]

[0088] In a further preferred embodiment, Z is selected from structures (Z21) to (Z38) and (Z38a) shown below. [ka]

[0089] Here, the connection to L is shown as a wavy bond. In structure (Z38), B (-) is an anion, preferably a pharma- ceutically acceptable anion. Ring Z is selected from structures (Za) to (Zm) as defined above, preferably structures (Za) to (Zj).

[0090] In preferred embodiments, Z comprises a (hetero)cyclooctene or (hetero)cycloheptene moiety, preferably according to structure (Z8), (Z26), (Z27), (Z28), or (Z37), or (Z38a), more preferably according to structure (Z8), (Z26), (Z27), (Z28), or (Z37), which is optionally substituted. Each of these preferred options for Z is further defined below.

[0091] Thus, in a preferred embodiment, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, a heterocycloheptyne moiety according to structure (Z37) is unsubstituted.

[0092] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably a (hetero)cyclooctene moiety according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z39) shown below, where V is (CH2) lwhere l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3, or 4, more preferably, l is 0, 1, or 2, and most preferably, l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z conforms to structure (Z42), further defined below.

[0093] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27), or (Z28), which are optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) shown below, where Y 1 is O or NR 11 where R 11 are independently hydrogen, linear or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring in (Z40) is optionally O-sulfonylated at one or more positions, while the ring in (Z41) can be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), which is further defined below.

[0094] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and conforms to the structure (Z37). [ka]

[0095] In particularly preferred embodiments, Z comprises a cyclooctenyl group and conforms to the structure (Z42). [ka] Where: -*The bond labeled with is connected to S, ** The wavy bond labeled with is connected to L, -R 15 are independently hydrogen, halogen, -OR 16 , NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 19 is hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24(hetero)arylalkyl groups, where the alkyl group is optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted or R 19 is a second occurrence of Z (or Q) or D connected via a spacer moiety, -l is an integer in the range of 0 to 10.

[0096] In a preferred embodiment of a group according to structure (Z42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of a group according to structure (Z42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of a group according to structure (Z42), R 19 is H. In preferred embodiments of groups according to structure (Z42), l is 0 or 1, and more preferably, l is 1.

[0097] In particularly preferred embodiments, Z comprises a (hetero)cyclooctynyl group and conforms to the structure (Z43). [ka] (Z43) Where: -* The bond labeled with is connected to S, ** The wavy bond labeled with is connected to L, -R15 are independently hydrogen, halogen, -OR 16 , NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y is N or CR 15 It is.

[0098] In a preferred embodiment of a group according to structure (Z43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of groups according to structure (Z43), Y is N or CH, more preferably Y=N.

[0099] In particularly preferred embodiments, Z comprises a heterocycloheptynyl group according to structure (Z37) or (Z38a), preferably according to structure (Z37), and ring Z is a triazole. [ka]

[0100] In an alternative preferred embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. the bond shown as - is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkanyl group or a cycloalkanyl group, preferably a cycloalkanyl group, where the (hetero)cycloalkanyl group is optionally substituted. Preferably, the (hetero)cycloalkanyl group is a (hetero)cyclopropanyl group, a (hetero)cyclobutanyl group, a norbornane group, a norbornene group, a (hetero)cycloheptanyl group, a (hetero)cyclooctanyl group, a (hetero)cyclononyl group, or a (hetero)cyclodecanyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropanyl groups, (hetero)cycloheptanyl groups, or (hetero)cyclooctanyl groups, where the (hetero)cyclopropanyl group, the trans-(hetero)cycloheptanyl group, or the (hetero)cyclooctanyl group are optionally substituted. Preferably, Z comprises a cyclopropanyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornane or norbornene group according to structure (Z46), a (hetero)cycloheptanyl moiety according to structure (Z47), or a (hetero)cyclooctanyl moiety according to structure (Z48), where Y 3 is C(R 23 )2, NR 23 or O, 23are individually hydrogen, C1-C6 alkyl, or are optionally connected to L via a spacer, and the bond labeled with -- is a single bond or a double bond. In a further preferred embodiment, the cyclopropanyl group conforms to structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group conforms to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group conforms to structure (Z52), (Z53), (Z54), (Z55) or (Z56). [ka]

[0101] wherein the R group(s) on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is typically selected from structures (Zn)-(Zu), where: ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z44) to (Z56) to which the ring Z is condensed, * The carbon labeled with is directly connected to the peptide chain of the antibody. Preferred rings Z are selected from (Zo) to (Zr). The connecting group Z is formed in the context of this embodiment by reaction with a (hetero)cycloalkene, so that the bond shown above as --- is a single bond. [ka]

[0102] In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably a nucleophilic substitution or a Michael addition, preferably a Michael addition. A preferred Michael reaction is a thiol-maleimide ligation, most preferably Q is a maleimide and F is a thiol group. Preferably, the thiol is present in the side chain of a cysteine ​​residue. In a preferred embodiment, the connecting group Z comprises a succinimidyl ring or a ring-opened succinamide derivative thereof. Preferred choices of the connecting group Z include moieties selected from (Z57) to (Z71) shown below. [ka]

[0103] Where: * The wavy bond(s) labeled with R are optionally connected to the antibody Ab via a linker, and the unlabeled wavy bond is optionally connected to the payload via a linker. 29 is C 1~12 Alkyl, preferably C 1~4 is alkyl, most preferably ethyl; X 1 is O or S, preferably X 1 =O. (Z67)~(Z71) ** The nitrogen atom labeled with corresponds to the nitrogen atom of the side chain of a lysine residue in the antibody. The carbon atoms of the phenyl groups of (Z69) and (Z70) are optionally substituted and preferably optionally fluorinated.

[0104] In a preferred embodiment, the connecting group Z comprises a moiety selected from (Z1)-(Z71).

[0105] Linker L The linker L connects the payload D with the connecting group Z (in a conjugate according to structure (1)) or the payload D with the reactive group Q (in a compound according to structure (2)). Linkers are known in the art and may be cleavable or non-cleavable. The linker L preferably comprises a self-immolative group or a cleavable linker comprising a peptide spacer and a para-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0106] In a preferred embodiment, (L 4 ) q is connected to the payload D, (L 1 ) n is connected to Z or Q, structure -(L 1 ) n- (L 2 ) o- (L 3 ) p- (L 4 ) q - as a linker L, where L 1 , L 2 , L 3 , and L 4 is a linker or linking unit, and each of n, o, p, and q is individually 0 or 1, and n+o+p+q is at least 1. In a preferred embodiment, at least the linker L 1 and L 2 is present (i.e., n=1, o=1, p=0 or 1, q=0 or 1), and more preferably, the linker L 1 , L 2 , and L 3 exists, and L 4 is present or absent (i.e., n=1, o=1, p=1, q=0 or 1). In one embodiment, the linker L 1 , L 2 , L 3 , and L 4 (i.e., n=1, o=1, p=1, q=1). In one embodiment, the linker L 1 , L 2 , and L 3 exists, and L 4does not exist (i.e., n=1, o=1, p=1, q=0).

[0107] Linkers, especially linker L 1 may contain one or more branching points for attaching multiple payloads to a single connecting group. In a preferred embodiment, the linker of the conjugate according to the invention comprises a branching moiety. A "branching moiety" in the context of the present invention refers to a moiety embedded in a linker that connects three moieties. In other words, the branching moiety comprises at least three bonds to other moieties, typically one bond to Z or Q, one bond to payload D, and one bond to a second payload D. The branching moiety, when present, is preferably located at the linker L 1 More preferably, Sp 2 Part of, or NR 13 embedded as a nitrogen atom of. Any moiety that contains at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. In a preferred embodiment, the branched moiety is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring, or a polycyclic moiety. Most preferably, the branched moiety is a nitrogen atom.

[0108] Linker L 1 Linker L 1 is absent (n=0) or present (n=1). 1 exists and n=1. L 1 For example, linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200The alkylene group may be selected from the group consisting of an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, an alkylarylene group, an arylalkylene group, an arylalkenylene group, and an arylalkynylene group. Optionally, the alkylene group, the alkenylene group, the alkynylene group, the cycloalkylene group, the cycloalkenylene group, the cycloalkynylene group, the alkylarylene group, the arylalkylene group, the arylalkenylene group, and the arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, which are preferably O, S(O), y’ , and N.R. 21 wherein y′ is 0, 1, or 2, preferably y′=2; R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups.

[0109] In a preferred embodiment, the linker L 1 contains a polar group. Such polar groups include (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,z'-diaminoalkanes (where z' is the number of carbon atoms in the alkane, preferably z'=1-10), -(O) a -C(O)-NH-S(O)2-NR 13 - (see structure (23) as further defined below), -C(S(O)3 (-) )-, -C(C(O)2 (-) )-, -S(O)2-, -P(O)2 (-) -, -O(CH2CH2O) t -, -NR 30 (CH2CH2NR 30 ) t -, as well as the following two structures: [ka]

[0110] The polar group may also preferably comprise an amino acid selected from Arg, Glu, Asp, Ser, and Thr, where a and R 13 is further defined below with respect to structure (23). t is an integer in the range of 0 to 15, preferably 1 to 10, more preferably 2 to 5, and most preferably t=2 or 4. Each R 30 are, respectively, H, C 1~12 Alkyl, C1~ 12 Aryl, C 1~12 Alkaryl or C 1~12 Aralkyl. Linker L 1 may contain two or more such polar groups, such as at least two polar groups. The polar groups may also be linked to linkers L, which branch the branching moieties as defined elsewhere. 1 Preferably, nitrogen or carbon atoms are used as branching moieties. In the branch, -O(CH2CH2O) t The presence of polar groups is particularly preferred.

[0111] In a preferred embodiment, the linker L 1 is or contains a sulfamide group, preferably a sulfamide group according to structure (23). [ka]

[0112] The wavy lines represent the remainder of the compound, typically Q and L. 2 , L 3 , L 4 or D, preferably Q and L 2 Preferably, (O) a The C(O) moiety is connected to Q and is 13 The part is L 2 , L 3 , L 4 or D, preferably L2 is connected to.

[0113] In structure (23), a=0 or 1, preferably a=1, R 13 is hydrogen, C 1~ C 24 Alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (Hetero)aryl groups, C 3~ C 24 Alkyl(hetero)aryl groups, and C 3~ C 24 (hetero)arylalkyl groups, 1~ C 24 Alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (Hetero)aryl groups, C 3~ C 24 Alkyl(hetero)aryl groups, and C 3~ C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 14 and R 14 are independently hydrogen and C 1~ C4 alkyl groups or R 13 is preferably linked via a spacer moiety to Sp 2 and in one embodiment, D is connected to N via -(B) e -(A) f -(B) g It is connected to N via -C(O)-.

[0114] In a preferred embodiment, R 13 is hydrogen or C1-C 20 More preferably, R 13 is hydrogen or C1-C 16 is an alkyl group, and even more preferably, R 13 is hydrogen or C1-C 10alkyl groups, which are optionally substituted, including O, S, and NR 14 R is optionally interrupted by one or more heteroatoms, preferably O, selected from 14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 13 is hydrogen. In another preferred embodiment, R 13 is C1~C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 is an alkyl group, optionally interrupted by one or more O atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is further preferred that R is a (poly)ethylene glycol chain containing a terminal -OH group. 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl, and i-propyl, even more preferably from the group consisting of hydrogen, methyl, and ethyl. Even more preferably, R 13 is hydrogen or methyl, and most preferably R 13 is hydrogen.

[0115] In a preferred embodiment, L 1 follows the structure (24). [ka]

[0116] where a and R 13 is as defined above, and Sp 1 and Sp 2 is independently a spacer moiety, and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, more preferably b=0 and c=1. In one embodiment, the spacer Sp 1and Sp 2 are independently linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene groups, and C9-C 200 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. When the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, the groups are preferably interrupted by one or more O atoms and / or one or more SS groups.

[0117] More preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C100 Alkynylene group, C3-C 100 Cycloalkylene group, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkyl arylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylalkenylene groups, and C9-C 100 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0118] Even more preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylene group, C3-C 50 Cycloalkylene group, C5-C 50 Cycloalkenylene group, C8-C 50 Cycloalkynylene group, C7-C 50 Alkyl arylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene groups, and C9-C 50and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0119] Even more preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylene group, C3-C 20 Cycloalkylene group, C5-C 20 Cycloalkenylene group, C8-C 20 Cycloalkynylene group, C7-C 20 Alkyl arylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene groups, and C9-C 20 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0120] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and include O, S, and NR 16 , optionally interrupted by one or more heteroatoms, preferably selected from the group of O, 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0121] Most preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 20 alkylene groups, which are optionally substituted, O, S, and NR 16 wherein R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is selected from the group consisting of O, S, and NR 16 , preferably O and / or SS, optionally interrupted by one or more heteroatoms, 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0122] Therefore, the preferred spacer moiety Sp 1 and Sp 2 is -(CH2) r -, -(CH2CH2) r -, -(CH2CH2O) r -, -(OCH2CH2) r -, -(CH2CH2O) r CH2CH2-, -CH2CH2(OCH2CH2) r -, -(CH2CH2CH2O) r -, -(OCH2CH2CH2) r -, -(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r In the formula, r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, 4, 5, 6, 7, or 8, even more preferably 1, 2, 3, 4, 5, or 6, and even more preferably 1, 2, 3, or 4.

[0123] Alternatively, the preferred linker L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein - d = 0 or 1, preferably d = 1, - e = 0 to 10, preferably e = an integer in the range of 0, 1, 2, 3, 4, 5, or 6, preferably an integer in the range of 1 to 10, most preferably e = 1, 2, 3, or 4; - f = 0 or 1, preferably f = 0, -d+e+f is at least 1, preferably in the range of 1 to 5, preferably d+f is at least 1, preferably d+f=1. - g = 0 or 1, preferably g = 1; - k = 0 or 1, preferably k = 1; -A is a sulfamide group according to structure (23), -B is a -CH2-CH2-O- or -O-CH2-CH2- moiety, or e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 a -CH2- moiety, where e1 is defined as e, -W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH2) m C(O)-, -C(O)(CH2) m C(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably W is -OC(O)NH-, -C(O)(CH2) m C(O)NH- or -C(O)NH-, where m is an integer in the range of 0 to 10, preferably m=0, 1, 2, 3, 4, 5 or 6, and most preferably m=2 or 3; Preferably, L 1 (W) k via Q, and (C(O)) g Preferably, L via C(O) 2 , L 3 , L 4 , or D, preferably L 2 is connected to.

[0124] In the context of this embodiment, the wavy line in structure (23) represents (W) k , (B) e , and (C(O)) g A preferably conforms to structure (23), where a=1 and R 13 =H or C1~C 20 Alkyl groups, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0125] Preferred linkers L 1 Structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein (a) k=0, d=1, g=1, f=0, B=-CH2-CH2-O-, and e=1, 2, 3, or 4, preferably e=2. (b) k=1, W=-C(O)(CH2) m C(O)NH-, m=2, d=0, (B) e =-(CH2-CH2-O) e1 -CH2-CH2-, f=0, g=1, e1=1, 2, 3, or 4, preferably e=1. (c) k=1, W=-OC(O)NH-, d=0, B=-CH2-CH2-O-, g=1, f=0, e=1, 2, 3, or 4, preferably e=2. (d) k = 1, W = -C(O)(CH2) m C(O)NH-, m=2, d=0, (B) e =-(CH2-CH2-O) e1 -CH2-CH2-, f=0, g=1, e1=1, 2, 3, or 4, preferably e1=4. (e)k=1, W=-OC(O)NH-, d=0, (B) e =-(CH2-CH2-O) e1 -CH2-CH2-, g=1, f=0, e1=1, 2, 3, or 4, preferably e1=4. (f)k=1, W=-(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, m=3, d=0, (B) e =-(CH2-CH2-O) e1 -CH2-CH2-, g=1, f=0, e1=1, 2, 3, or 4, preferably e1=4. (g) k=0, d=0, g=1, f=0, B=-CH2-CH2-O-, and e=1, 2, 3, or 4, preferably e=2. (h) k=1, W=-C(O)NH-, d=0, g=1, f=0, B=-CH2-CH2-O-, e=1, 2, 3, or 4, preferably e=2.

[0126] In a preferred embodiment, the linker L 1 contains a branched nitrogen atom, which is a group consisting of Q or Z and (L 2 ) o and contains as a substituent a further moiety D, which is preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR 13 where R 13 is connected to the second occurrence of D via a spacer moiety. Alternatively, the branched nitrogen atom can be represented by the structure -(W) k -(A) d -(B) e -(A) f -(C(O)) g -Following L 1 In one embodiment, L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -N * [-(A) d -(B) e -(A) f -(C(O)) g’ -]2, in which A, B, W, d, e, f, g, and k are as defined above and are individually selected for each occurrence; N * is -(A) d -(B) e -(A) f -(C(O)) g’ - are two examples of connected branched nitrogen atoms, where both (C(O)) g’ The part is -(L 2 ) o -(L 3 ) p -(L 4 ) q -D, wherein L 2 , L 3 , L 4, o, p, q, and D are as defined above and are each individually selected. In a preferred embodiment, L 2 , L 3 , L 4 , o, p, q, and D are each (C(O)) g is the same for both parts connected to

[0127] Preferred linkers L containing a branched nitrogen atom 1 Structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -N * [-(A') d’ -(B') e’ -(A') f’ -(C(O)) g” -]2, wherein (i) k=d=g=e'=1, f=d'=g'=0, W=-C(O)-, B=B'=-CH2-CH2-O-, A is according to structure (23), a=0 and R 13 =H, and e=1, 2, 3, or 4, preferably e=2. (j) k=d=g=e'=g'=1, f=d'=0, W=-C(O)-, B=B'=-CH2-CH2-O-, A is according to structure (23), a=0 and R 13 =H, and e=1, 2, 3, or 4, preferably e=2.

[0128] Linker L 2 Linker L 2 is absent (o=0) or present (o=1). 2 is present and o=1. 2 is a peptide spacer. The peptide spacer is preferably (NH-CR 17 -CO) n wherein R 17represents an amino acid side chain as known in the art. As used herein, the amino acids may be natural or synthetic amino acids. Preferably, the amino acid(s) are all in their L-configuration. n is an integer in the range of 1 to 5, preferably in the range of 2 to 5. Thus, the peptide spacer preferably comprises 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3) or tetrapeptide (n=4), most preferably the peptide spacer is a dipeptide. Although any peptide spacer may be used, preferably the peptide spacer is Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys , Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Gly-Gly-Phe-Gly and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn, most preferably Val-Cit or Val-Ala. As used herein, AcLys is acetyl lysine and iGlu is isoglutamate. In one embodiment, L 2 In one embodiment, L 2 =Val-Ala.

[0129] R 17represents an amino acid side chain preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyl lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, more preferably from the side chains of Val, Cit, Ala, Glu, and Lys. Alternatively, R 17 is preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp), and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred embodiments of R are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). 17 is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0130] In a particularly preferred embodiment, the peptide spacer may be represented by the general structure (L3): [ka]

[0131] In the formula, R 17is as defined above, preferably R 17 is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates (L 1 ) n and (L 3 ) p and preferably according to the structure (L3). 2 is via NH (L 1 ) n through C(O) (L 3 ) p is connected to.

[0132] Linker L 3 Linker L 3 is absent (p=0) or present (p=1). 3 is present and p=1. 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is a paraaminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L4). [ka]

[0133] Here, the wavy lines represent Q or Z, L 1 Or L 2 , and L 4 or D. Typically, the PABC derivatives are connected to Q, Z, L, or D via NH. 1 , or L 2 , preferably L 2 to, and through O to L 4 Or connected to D.

[0134] A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Preferred 5-membered rings are oxazole, thiazole and furan. Preferred 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,4-phenyl, 2,5-pyridyl or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0135] R 21 , H, R 26 , C(O)OH, and C(O)R 26 Selected from R 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups, and C3-C 10 (hetero)arylalkyl groups, which are optionally substituted, include O, S, and NR 28 and R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably -(CH2CH2O) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3, or 4. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0136] Linker L 4 Linker L 4 is absent (q=0) or present (q=1). 4 is present and q=1. 4 teeth, -Structure-NR 22 -(C z -alkylene)-C(O)-, where z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl; -Structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 an ethylene glycol spacer according to -C(O)-, where e6 is an integer ranging from 1 to 10, e7 is an integer ranging from 1 to 3, and R 22 is H or C1-C4 alkyl, and -Structure-NR 22 -(C z -alkylene)-NR 22 -(C(O)) h - a diamine spacer according to the formula (wherein h is 0 or 1, z is an integer in the range of 1 to 20, and R 22 is H or C1-C4 alkyl.

[0137] Linker L 4 represents an aminoalkanoic acid spacer, i.e., -NR 22 -(C z -alkylene)-C(O)-, where z is an integer in the range of 1 to 20, preferably 1 to 10, and most preferably 1 to 6. As used herein, the aminoalkanoic acid spacer is typically linked to L via a nitrogen atom. 3 and to D via a carbonyl moiety. 4 is selected from 6-aminohexanoic acid (Ahx, z=5), β-alanine (z=2) and glycine (Gly, z=1), and even more preferably is 6-aminohexanoic acid or glycine.4= 6-aminohexanoic acid. In one embodiment, L 4 = glycine. 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0138] Alternatively, the linker L 4 is the structure -NR 22- (CH2-CH2-O) e6- (CH2) e7- (C(O)-, where e6 is an integer in the range of 1 to 10, preferably e6 is in the range of 2 to 6, and e7 is an integer in the range of 1 to 3, preferably e7 is 2. In the present specification, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0139] Alternatively, the linker L 4 is the structure -NR 22 -(C z -alkylene)-NR 22 -(C(O)) h -, where h is 0 or 1, z is an integer in the range of 1 to 20, preferably an integer in the range of 2 to 6, even more preferably z=2 or 5, and most preferably z=2. 22 is H or C1-C4 alkyl. 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is methyl. As used herein, h is preferably 1, in which case the linker L 4 is particularly suitable for conjugation via the phenolic hydroxyl groups present on the payload D.

[0140] Payload D D represents a targeting molecule D that is or will be attached to the antibody, also referred to in the art as a payload. D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof. Alternatively, D is defined as a pharma- ceutically active substance, such as an anti-cancer drug, preferably a cytotoxin. Preferably, D is selected from the group consisting of anthracyclines, camptothecins, maytansinoids, enediynes, amanitins, auristatins, and pyrrolobenzodiazepine dimers, more preferably D is selected from the group consisting of enediynes, auristatins, and camptothecins. 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.

[0141] In a preferred embodiment, the enediyne is selected from calicheamicin, esperamicin, shishijimicin, and namenamemycin, 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 compounds shown in Figure 6, preferably selected from the group consisting of topotecan, ciratecan, cositecan, exatecan, exatecan-S, DXd, SN-38, lurtotecan, gimatecan, belotecan, rubitecan, AMDCPT, and G-AMDCPT, more preferably selected from the group consisting of exatecan or DXd, and most preferably selected from the group consisting of exatecan.

[0142] In particularly preferred embodiments, D is selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably, D is exatecan.

[0143] Compounds according to general structure (2) may contain two or more moieties D. When two or more cytotoxins D are present, the cytotoxins D may be the same or different, typically they are the same. In a preferred embodiment, compounds according to general structure (2) contain one or two occurrences of D, most preferably two occurrences of D. Typically, the second occurrence of D is present in a linker L, which may contain a branched moiety, typically a branched nitrogen atom, connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branched moiety via the same linker. Similarly, antibody conjugates according to structure (1) may contain two or more moieties D per connecting group Z.

[0144] Preferred Antibody Conjugates A preferred antibody conjugate according to the first aspect is selected from the group consisting of compounds (I) to (III), more preferably (II) or (III), most preferably (II). A more preferred antibody conjugate is selected from (X) to (XIII). In one particularly preferred embodiment, the antibody conjugate is selected from (Xa), (XIb), (XIIg), (XIIh), and (XIIIe). In an even more preferred embodiment, the antibody conjugate is selected from (XI) and (XIII), more preferably (XIb) or (XIIIe), more preferably the antibody conjugate is according to (XIII), most preferably (XIIIe). The structures of these antibody conjugates are defined below.

[0145] The antibody conjugate (I) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L3 )-(L 4 ) q -D} x ] y (I) During the ceremony, -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined above as -(A) d -(B) e -(A) f -(C(O)) g - is a linker represented by -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), -L 4 is -N-(C z -alkylene)-C(O)- or -NR 22 -(C z -alkylene)-NR 22 where z and R 22 is as defined above, -q=0 or 1.

[0146] In the context of the antibody conjugate (I), L 1 For d=1 (A according to structure (23), a=1 and R 13 =H), e=2, f=0 and g=1. In the context of the antibody conjugate (I), L 2 In the context of the antibody conjugate (I), L 3 For R 21 = H. In the context of antibody conjugate (I), when q=1, it is preferred that z=1 or 5.

[0147] In a particularly preferred embodiment, the antibody conjugate according to structure (I) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0148] The antibody conjugate (II) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-D} x ] y (II) During the ceremony, -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), where R 21 =H.

[0149] In the context of the antibody conjugate (II), L according to structure (23) 1 , e=2, for A, a=1 and R 13 In the context of the antibody conjugate (II), L 2 =Val-Cit is preferred.

[0150] In a particularly preferred embodiment, the antibody conjugate according to structure (II) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0151] The antibody conjugate (III) has the following structure: AB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-(L 4 )-D} x ] y (III) Where: -AB, L 6 , Z, D, x and y are as defined above; -L 1 is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 is Val-Cit or Val-Ala, -L 3 is a PABC derivative according to structure (L4), where R 21 =H, -L 4 -NR 22 -(C z -alkylene)-NR 22 -, wherein R 22 is as defined above, and z is an integer in the range of 1 to 6.

[0152] In the context of the antibody conjugate (III), L 1 For e=2, a=1 and R 13 In the context of the antibody conjugate (III), L 2In the context of the antibody conjugate (III), it is preferred that z=2.

[0153] In a particularly preferred embodiment, the antibody conjugate according to structure (III) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0154] The antibody conjugate (X) has a linker-payload portion according to the following structure: [ka] During the ceremony, - The wavy line indicates the connection to Z, -L 2 , o and D are as defined above.

[0155] L 2 may be absent, preferably L 2 is present and o=1. For preferred antibody conjugates (Xa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (Xb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (X) preferably has the structure (Xa).

[0156] In a particularly preferred embodiment, the antibody conjugate according to structure (X) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0157] The antibody conjugate (XI) has a linker-payload portion according to the following structure: [ka] Where: - The wavy line indicates the connection to Z, -L 2 , o and D are as defined above.

[0158] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. The antibody conjugate (XI) preferably has the structure (XIb).

[0159] In a particularly preferred embodiment, the antibody conjugate according to structure (XI) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably calicheamicin as payload D.

[0160] The antibody conjugate (XII) has a linker-payload portion according to the following structure: [ka] During the ceremony, - The wavy line indicates the connection to Z, -L 2 , L 4 , o, q, and D are as defined above.

[0161] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2.

[0162] L 4 For the preferred antibody conjugate (XIIc), q=0 and L 4 For the preferred antibody conjugate (XIId), q=1 and L 4 Structure -NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0163] For the preferred antibody conjugate (XIIe), 2 follows the structure (L3), R 17 is CH3, q=0, and L 4 For the preferred antibody conjugate (XIIf),2 follows the structure (L3), R 17 is CH3, q=1, and L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, where z is an integer ranging from 1 to 10, preferably z=2, and R 22 is H or C1-C4 alkyl.

[0164] For the preferred antibody conjugate (XIIg), 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0, L 4 For the preferred antibody conjugate (XIIh), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0165] In the context of the antibody conjugate (XII), z is preferably an integer in the range of 1 to 10, more preferably z=2 to 6, and most preferably z=2. In the context of the antibody conjugate (XII), R 22 is more preferably H or CH3.

[0166] In the context of the antibody conjugate (XII), structures (XIIg) and (XIIh) are most preferred.

[0167] In a particularly preferred embodiment, the antibody conjugate according to structure (XII) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0168] The antibody conjugate (XIII) has a linker-payload portion according to the following structure: [ka] During the ceremony, - The wavy line indicates the connection to Z, -L 2 , L 4 , o, q, and D are as defined above.

[0169] L 2 may be absent, preferably L 2 is present and o=1. For the preferred antibody conjugate (XIIIa), L 2 follows the structure (L3), R 17 For the preferred antibody conjugate (XIIIb), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH3.

[0170] L 4 For the preferred antibody conjugate (XIIIc), q=0 and L 4 For the preferred antibody conjugate (XIIId), q=1 and L 4 Structure -NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R22 is H or C1-C4 alkyl.

[0171] For the preferred antibody conjugate (XIIIe), 2 follows the structure (L3), R 17 is CH3, q=0, and L 4 For the preferred antibody conjugate (XIIIf), L 2 follows the structure (L3), R 17 is CH3, q=1, and L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, where z is an integer in the range of 1 to 10, preferably z=2, and R 22 is H or C1-C4 alkyl.

[0172] For the preferred antibody conjugate (XIIIg), 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=0, L 4 For the preferred antibody conjugate (XIIIh), L 2 follows the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure - NR 22 -(C z -alkylene)-NR 22 -, wherein z is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl.

[0173] In the context of the antibody conjugate (XIII), z is preferably an integer in the range of 1 to 10, more preferably z=2 to 6, and most preferably z=2. In the context of the antibody conjugate (XIII), R 22 is more preferably H or CH3.

[0174] In the context of the antibody conjugate (XIII), the structure (XIIIe) is most preferred.

[0175] In a particularly preferred embodiment, the antibody conjugate according to structure (XIII) comprises payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan, and DXd, more preferably payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably exatecan as payload D.

[0176] In one particularly preferred embodiment, the antibody conjugate according to the invention conforms to structure (XIb) as defined above, wherein the antibody is cofetuzumab as defined above and the payload is calicheamicin.

[0177] In one particularly preferred embodiment, the antibody conjugate according to the invention conforms to structure (XIIIe) defined above, the antibody is cofetuzumab as defined above and the payload is exatecan.

[0178] These preferred antibody conjugates are further preferably conjugated via a glycan (i.e. b=1), more preferably a trimmed glycan (i.e. j=0). It is further preferred herein that S=GalNAc and w'=0. It is further preferred herein that the connecting group Z is formed by azide-alkyne cycloaddition, preferably the connecting group Z=(Z39), with ring Z=(Za) and V=CH2. It is further preferred herein that x=1. It is further preferred herein that y=2, more preferably x=1 and y=2.

[0179] In a most preferred embodiment, the antibody conjugate according to the invention conforms to structure (XIIIe) defined above, wherein the antibody is enfortumab YTE as defined above, the payload is exatecan, where b=1, e=0, S=GalNAc, w'=0, connecting group Z=(Z39), ring Z=(Za) and V=CH2, x=1 and y=2.

[0180] Compounds conforming to general structure (2) The compound has the general structure (2): QLD (2) During the ceremony, -Q is a reactive moiety, - L is a linker connecting Z to D, D is selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof;

[0181] Compounds of general structure (2) may also be referred to as "linker-drug constructs" to include the linker L and payload D of the final conjugate. Compounds of general formula (2) may be prepared by those skilled in the art using standard organic synthesis techniques and as illustrated in the Examples. Linker L and payload D are defined above in the context of the conjugate according to structure (1).

[0182] Reactive moiety Q Compounds according to general structure (2) include a reactive moiety Q. In the context of the present invention, the term "reactive moiety" can refer to a chemical moiety that includes a reactive group, but also to the reactive group itself. For example, a cyclooctynyl group is a reactive group that includes a CC triple bond as a reactive group. Similarly, an N-maleimidyl group is a reactive group that includes a CC double bond as a reactive group. However, reactive groups, such as an azide reactive group, a thiol reactive group, or an alkynyl reactive group, can also be referred to as reactive moieties herein.

[0183] Q is S(F) x In other words, Q is reactive to and complementary to F. As used herein, a reactive group is referred to as "complementary" to a reactive group when the reactive group selectively reacts with the reactive group, optionally in the presence of another functional group. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Thus, compounds according to general structure (2) are advantageously used in conjugation reactions in which a chemical reaction between Q and F occurs, thereby forming an antibody conjugate comprising a covalent connection between the payload D and the antibody.

[0184] The exact nature of Q and F depends on the type of conjugation reaction used. The skilled person will be able to select the appropriate combination of Q and F. Preferably, Q, and therefore also F, are reactive in a cycloaddition or nucleophilic reaction. Thus, Q preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety, more preferably Q is a click probe, a thiol-reactive moiety, or an amine-reactive moiety, and most preferably Q is a click probe. The click probe is reactive in a cycloaddition (click reaction), and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an alkene or alkyne moiety, more preferably the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne. Exemplary thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, alenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, vinylsulfones, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Exemplary amine-reactive moieties are selected from N-hydroxysuccinimidyl esters and other activated esters, p-nitrophenyl carbonate and other activated carbonates, isocyanates, isothiocyanates, haloacetamides, and benzyl halides. In a preferred embodiment, Q is selected from an alkene moiety, an alkyne moiety, a thiol-reactive moiety or an amine-reactive moiety, more preferably an alkene moiety or an alkyne moiety, even more preferably an alkyne moiety. In the present specification, the alkene is preferably a (hetero)cycloalkene, and the alkyne is preferably a terminal alkyne or (hetero)cycloalkyne. Most preferably, Q is a cyclic (hetero)alkyne moiety. Each of these moieties is further defined below.

[0185] Thus, in a particularly preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, and the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group or a (hetero)cyclodecynyl group. In the present specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, and the (hetero)cyclooctynyl group is optionally substituted.

[0186] In particularly preferred embodiments, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and conforms to the structure (Q1). [ka] Where: Bonds shown as ---- are double or triple bonds, -R 15 are independently hydrogen, halogen, -OR 16 , NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 , or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by (-) is an anion, and each R 31 are individually, R 15 or a connection to D connected via L, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; -v is an integer in the range of 0 to 16.

[0187] Typically, v = (u + u') × 2 (the connection to L shown by the wavy coupling is Y 2 (when the connection to L represented by the wavy bond is via one of the carbon atoms) or [(u+u')×2]−1 (when the connection to L represented by the wavy bond is via one of the carbon atoms).

[0188] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and conforms to structure (Q1a). [ka] Where: -R 15 and Y2 is as defined above, - u is 0, 1, 2, 3, 4 or 5, -u' is 0, 1, 2, 3, 4, or 5, and u+u'=4, 5, 6, 7, or 8; -v is an integer in the range of 8 to 16.

[0189] In preferred embodiments, u+u'=4, 5, or 6, more preferably u+u'=5. In preferred embodiments, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0190] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2)-(Q20) and (Q20a) shown below. [ka]

[0191] Here, the connection to L, shown as a wavy bond, may be to any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) may have a connection to L or may contain a hydrogen atom or may be optionally functionalized. (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) , or I (-) and most preferably selected from B (-) teeth (-) In the conjugation reaction, B (-) In any case, B will exchange with anions present in the reaction mixture. (-) does not have to be a pharma- ceutically acceptable anion. When (Q19) is used for Q, the negatively charged counterion is preferably pharma- ceutically acceptable upon isolation of the conjugate according to the invention, so that the conjugate can be readily used as a drug.

[0192] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38) and (Q38a) shown below. [ka]

[0193] In structure (Q38), B (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably selected from B (-) teeth (-) It is OTf.

[0194] In preferred embodiments, Q comprises a (hetero)cyclooctyne or (hetero)cycloheptyne moiety, preferably according to structure (Q8), (Q26), (Q27), (Q28), (Q37), or (Q38a), more preferably according to structure (Q8), (Q26), (Q27), (Q28), or (Q37), which is optionally substituted. Each of these preferred options for Q is further defined below.

[0195] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also referred to as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is unsubstituted.

[0196] In an alternative preferred embodiment, Q comprises a cyclooctyne moiety according to structure (Q8), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), more preferably a cyclooctyne moiety according to (Q29), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q39) shown below, where V is (CH2) l where l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3, or 4, more preferably, l is 0, 1, or 2, and most preferably, l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q conforms to structure (Q42), further defined below.

[0197] In alternative preferred embodiments, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27), or (Q28), also referred to as a DIBO, DIBAC, DBCO, or ADIBO group, which is optionally substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) shown below, where Y 1 is O or NR 11 where R 11 are independently hydrogen, linear or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring in (Q40) may be optionally O-sulfonylated at one or more positions, while the ring in (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), further defined below.

[0198] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and conforms to the structure (Q37). [ka]

[0199] In particularly preferred embodiments, Q comprises a cyclooctynyl group and conforms to the structure (Q42). [ka] Where: -R 15 are independently hydrogen, halogen, -OR 16 , NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -R 19is hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group is optionally interrupted by one of a plurality of heteroatoms selected from the group consisting of O, N and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted or R 19 is a second occurrence of Q or D connected via a spacer moiety, -l is an integer in the range of 0 to 10.

[0200] In a preferred embodiment of the reactive group according to structure (Q42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 is H. In preferred embodiments of the reactive group according to structure (Q42), l is 0 or 1, more preferably, l is 1.

[0201] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and conforms to the structure (Q43). [ka] Where: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, where the alkyl group, the (hetero)aryl group, the alkyl(hetero)aryl group, and the (hetero)arylalkyl group are optionally substituted, and two substituents R 15 are linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero) substituent; R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 (hetero)arylalkyl groups, -Y is N or CR 15 It is.

[0202] In a preferred embodiment of the reactive group according to structure (Q43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of the reactive group according to structure (Q43), Y is N or CH, more preferably Y=N.

[0203] In particularly preferred embodiments, Q comprises a heterocycloheptynyl group and conforms to structure (Q37) or (Q38a), preferably structure (Q37). [ka]

[0204] In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, where the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, or trans-(hetero)cyclooctenyl groups, which are optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48), where Y 3 is C(R 23 )2, NR 23 or O, 23are individually hydrogen, C1-C6 alkyl, or are optionally connected to L via a spacer, and the bond labeled with --- is a single bond or a double bond. In a further preferred embodiment, the cyclopropenyl group conforms to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group conforms to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group conforms to structure (Q52), (Q53), (Q54), (Q55) or (Q56). [ka]

[0205] Here, the R group(s) on Si in (Q50) and (Q51) are typically alkyl or aryl, preferably C1-C6 alkyl.

[0206] In an alternative preferred embodiment, Q is a thiol-reactive probe. In this embodiment, Q is a reactive group that is compatible with cysteine ​​conjugation. Such probes are known in the art and can be selected from the group consisting of maleimide moiety, haloacetamide moiety, alenamide moiety, phosphonamidite moiety, cyanoethynyl moiety, vinylsulfone, vinylpyridine moiety, or methylsulfonylphenyloxadiazole moiety. Most preferably, Q comprises or is a maleimide moiety. The reagent can be mono-alkylated or can be a crosslinker for reaction with two cysteine ​​side chains.

[0207] In a further preferred embodiment, probe Q is selected from the group consisting of (Q57) to (Q71) shown below. [ka] Where: -X 6is H, halogen, PhS, MeS, preferably halogen such as Cl, Br, I, -X 7 is a halogen, such as PhS, MeS, preferably Cl, Br, I, etc. -R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 is alkyl, -R 25 , H, C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkaryl or C 1~12 aralkyl, preferably H or paramethylphenyl; wherein the aromatic rings in (Q61) and (Q63) may optionally be heteroaromatic rings, such as phenyl or pyridine rings.

[0208] In a preferred embodiment of the thiol-reactive probe (Q57), probe Q is selected from the group consisting of (Q72) to (Q74) shown below. [ka] Where: -R 27 is C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkaryl or C 1~12 It is aralkyl, -t is an integer in the range of 0 to 15, preferably 1 to 10.

[0209] In an alternative preferred embodiment, Q is an amine-reactive probe. In this embodiment, Q is a reactive group compatible with lysine conjugation. Such probes are known in the art and can be selected from the group consisting of N-hydroxysuccinimidyl groups, isocyanate groups, isothiocyanate groups, and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimidyl ester or p-nitrophenyl carbonate moiety.

[0210] In a further preferred embodiment, probe Q is selected from the group consisting of (Q75) to (Q79) shown below. [ka] Where X 2 is a halogen, preferably F.

[0211] In a preferred embodiment, Q is selected from the group consisting of (Q1) to (Q79).

[0212] Antibodies conforming to general structure (3) The antibody has the general structure (3): AB-[(L 6 ) b -{F} x ] y (3) During the ceremony, -AB is an antibody capable of targeting PTK7-expressing tumors, -b is 0 or 1, -L 6 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, in which G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1, or 2, and L7 is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-, -F is a reactive moiety, -x is 1 or 2, -y is 1, 2, 3, or 4.

[0213] The antibody of general structure (3) may also be referred to as a "(modified) antibody" since it is an antibody comprising a reactive group F, which is naturally occurring or the antibody has been modified to incorporate a reactive group F. The (modified) antibody according to general formula (2) may be prepared by one of skill in the art using standard organic and / or enzymatic synthesis techniques and as illustrated in the examples. 6 , b, x and y are defined above in the context of the conjugate according to structure (1).

[0214] Reactive moiety F F is reactive to Q in a conjugation reaction defined below, preferably the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. As the skilled person will appreciate, the options for F are the same as the options for Q, except that F and Q are reactive to each other. Thus, F preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety, more preferably F is a click probe, a thiol or an amine, and most preferably F is a click probe. The click probe is reactive in a cycloaddition (click reaction), and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an orthoquinone, a dioxothiophene, a sydnone, an alkene moiety, and an alkyne moiety. Preferably, the click probe comprises or is an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an orthoquinone, a dioxothiophene, or a sydnone, most preferably an azide. Exemplary thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, alenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, orthoquinone moieties, vinylsulfones, vinylpyridines, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Exemplary amine-reactive moieties are selected from N-hydroxysuccinimidyl esters, isocyanates, isothiocyanates, and benzyl halides. In a preferred embodiment, F is a click probe or thiol, more preferably F is an azide or thiol, and most preferably F is an azide.

[0215] Two or more reactive groups F may be present in the antibody. The reactive groups F in the antibody may be naturally occurring or may be placed in the antibody by certain techniques, such as (bio)chemical or genetic techniques. The reactive groups placed in the antibody are prepared by chemical synthesis, such as azides or terminal alkynes. Methods for preparing modified antibodies are known in the art, for example from WO2014 / 065661, WO2016 / 170186 and WO2016 / 053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between modified antibodies and linker-drug constructs is known to those skilled in the art.

[0216] Preferably, F is a click probe reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and sydnones. Preferred structures of the reactive group are structures (F1) to (F10) shown below. [ka]

[0217] Here, the wavy bonds represent connections to the payload. For (F3), (F4), (F8), and (F9), the payload can be connected to any one of the wavy bonds. The other wavy bonds can then be connected to hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups, and C1-C 24 a sulfonyl group, each of which (except hydrogen) may be optionally substituted, and optionally O, S, and NR 32 and R32 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. One of ordinary skill in the art will understand which R groups may apply to each of the groups F. For example, the R group attached to the nitrogen atom of (F3) may be selected from alkyl and aryl, and the R group attached to the carbon atom of (F3) may be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl. Preferably, the reactive moiety F is selected from azide or tetrazine. Most preferably, the reactive moiety F is azide.

[0218] In a second preferred embodiment, F is a thiol or a precursor thereof. The thiol or a precursor thereof F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive to a thiol-reactive probe Q in a thiol ligation. The thiol is preferably the thiol of the side chain of a cysteine ​​amino acid naturally occurring in the antibody AB, in which case the linker L 6 is absent (b=0), but optionally includes a linker L 6 Thiol precursors in the context of bioconjugation are known in the art and include disulfides which may be naturally occurring disulfide bridges present in antibodies, or synthetically introduced disulfides which are reduced as known in the art. Preferably, F is a thiol group of a cysteine ​​side chain.

[0219] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or a precursor thereof F is used in a conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive to the amine-reactive probe Q in a nucleophilic displacement. The amine is typically a primary amine, preferably the amine of the side chain of a lysine amino acid naturally occurring in the antibody AB, in which case the linker L 6 is absent (b=0), but optionally includes a linker L 6Preferably, F is a primary amine group of a lysine side chain.

[0220] Process for synthesizing antibody conjugates according to general structure (1) In a further aspect, the present invention relates to a process for the preparation of an antibody conjugate according to the invention, which process comprises a step of reacting Q of a compound according to the invention with a reactive group F of an antibody. Compounds according to general structure (2) and preferred embodiments thereof are described in more detail above. The process is carried out under conditions such that Q reacts with F to covalently link the antibody AB (3) to the payload D. In the method according to the invention, Q reacts with F to form a covalent bond between the antibody and the compound according to the invention. Complementary reactive groups Q and reactive groups F are known to the skilled person and are described in more detail below.

[0221] Any conjugation technique known in the art can be used to prepare the multifunctional antibody construct of the present invention.Suitable conjugation techniques include thiol ligation, lysine ligation, cycloaddition (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cycloaddition, strain-promoted quinone-alkyne cycloaddition).Preferred conjugation techniques used in the context of the present invention include nucleophilic reaction and cycloaddition, preferably, cycloaddition is [4+2] cycloaddition or [3+2] cycloaddition, and nucleophilic reaction is Michael addition or nucleophilic substitution. Suitable conjugation techniques are disclosed, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), WO2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, PCT / EP2021 / 050594, PCT / EP2021 / 050598 and NL2026947.

[0222] Thus, in a preferred embodiment of the conjugation process according to the invention, the conjugation is achieved via a nucleophilic reaction, such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is the acylation of a primary amino group with an activated ester. A preferred Michael reaction is the maleimide-thiol reaction, which is widely used in bioconjugation.

[0223] Thus, in a preferred embodiment of the conjugation process according to the present invention, the conjugation is achieved via cycloaddition. A preferred cycloaddition is a (4+2)-cycloaddition (e.g., Diels-Alder reaction) or a (3+2)-cycloaddition (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art and the skilled person knows how to carry them out.

[0224] The process according to this embodiment preferably relates to a Click reaction, more preferably a 1,3-dipolar cycloaddition, most preferably an alkyne / azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azide group. Click reactions such as 1,3-dipolar cycloadditions are known in the art and the skilled person will know how to carry them out.

[0225] Thus, the process for preparing an antibody conjugate according to the invention according to this embodiment comprises reacting a modified antibody of structure (3) with a compound according to structure (2) to obtain an antibody conjugate of structure (1).

[0226] In a preferred embodiment, the process for preparing an antibody-conjugate according to the invention comprises the steps of: (i) reacting an antibody containing y core N-acetylglucosamine (GlcNAc) moiety (where y=1, 2, 3, or 4) with an antibody of formula S(F) in the presence of a catalyst x -P(in the formula, S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside mono- or diphosphate, and the catalyst is S(F) x (which can transfer a moiety to the core-GlcNAc moiety) AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) (In the formula, -AB is an antibody capable of targeting PTK7-expressing tumors, -Fuc is fucose, - obtaining a modified antibody according to (ii) coupling the modified antibody to a compound represented by the structure (2): QLD (2) (In the formula, -Q is a reactive moiety, - L is a linker connecting Z to D, -D is reacted with a compound according to the formula (I) selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; and obtaining an antibody conjugate according to structure (1).

[0227] Step (i) In step (i), an antibody containing one, two, three, or four core N-acetylglucosamine moieties is reacted with an antibody of formula S(F) in the presence of a catalyst.x -P, wherein S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside monophosphate or diphosphate, and the catalyst is S(F) x The moiety can be transferred to a core-GlcNAc moiety. As used herein, the antibody is typically an antibody that has been trimmed to a core-GlcNAc residue, as further described below. Step (i) provides a modified antibody according to formula (26).

[0228] The starting material, i.e., an antibody comprising a core-GlcNAc substitution, is known in the art and can be prepared by known methods by the skilled artisan. In one embodiment, the process according to the invention further comprises deglycosylating the antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substitution, the core N-acetylglucosamine and the core N-acetylglucosamine substitution being optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH, and / or combinations thereof, the selection of which depends on the nature of the glycan. EndoSH is described in PCT / EP2017 / 052792, which is incorporated by reference herein, see Examples 1-3 and Sequence ID No. 1.

[0229] The structural features S and x are defined above for the antibody-conjugates according to the invention and apply equally to this embodiment. x The formula S(F) to be connected to xCompounds of -P are known in the art, e.g., Wang et al., Chem. Eur. J. 2010, 16, 13343-13345; Piller et al., ACS Chem. Biol. 2012, 7, 753; Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462 and WO2009 / 102820, all of which are incorporated herein by reference, but some compounds S(F) x In a preferred embodiment, S(F)-P and their synthesis are disclosed. x The nucleoside monophosphate or diphosphate P in -P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP), 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-(buta-3-amino acid)-2-deoxy-galactose-UDP. Most preferably, S(F) x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0230] SCIENCE FICTION) x Suitable catalysts capable of transferring a moiety to a core-GlcNAc moiety are known in the art. Suitable catalysts are those that are capable of transferring a moiety to a core-GlcNAc moiety, such as a specific sugar derivative nucleotide S(F) in that particular process. x-P is a catalyst for which the substrate. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcT) and β(1,4)-galactosyltransferases (GalT), and most preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases having a mutated catalytic domain. Suitable catalysts and mutations thereof are disclosed in WO2014 / 065661, WO2016 / 022027, and WO2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. The mutant enzymes described in WO2016 / 022027 and WO2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts are capable of recognizing internal sugars and sugar derivatives as acceptors. Thus, the sugar derivative S(F) x is linked to the core GlcNAc substituent in step (i), regardless of whether that GlcNAc is fucosylated or not.

[0231] 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.

[0232] Step (ii) In step (ii), the modified antibody is reacted with a compound according to general structure (2) comprising a reactive group F and a reactive group Q capable of reacting with a payload D to obtain an antibody conjugate according to structure (1) comprising a linking group Z resulting from the reaction between Q and F. Such reaction occurs under conditions such that the reactive group Q reacts with the reactive group F of the biomolecule to covalently link the antibody to the compound according to general structure (2). Step (ii) may also be referred to as a conjugation reaction.

[0233] In a preferred embodiment, in step (ii), the azide on the azide-modified antibody reacts with an alkynyl group, preferably a terminal alkynyl group, or a (hetero)cycloalkynyl group of a compound according to general structure (2), via a cycloaddition reaction. This cycloaddition reaction between a molecule containing an azide and a molecule containing a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the reactions known in the art as "click chemistry". In the case of a linker conjugate containing a terminal alkynyl group, the cycloaddition reaction needs to be carried out in the presence of a suitable catalyst, preferably a Cu(I) catalyst. However, in a preferred embodiment, the linker conjugate contains a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required and the reaction can occur spontaneously by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry."

[0234] application The present invention relates to a method of treating a subject in need of treatment, comprising administering an antibody conjugate according to the invention as defined above. The subject in need of treatment is typically a cancer patient. The use of antibody conjugates, such as antibody drug conjugates, is well known in the field of cancer treatment, and the antibody conjugate according to the invention is particularly suitable in this respect. The described method is typically suitable for the treatment of cancer. In the method according to this aspect, the antibody conjugate is typically administered in a therapeutically effective amount. This aspect of the invention can also be expressed as an antibody conjugate according to the invention for use in the treatment of a subject in need of treatment, preferably for the treatment of cancer. In other words, this aspect relates to the use of an antibody conjugate according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of a subject in need of treatment, preferably for the treatment of cancer. In this context, it is envisaged that the treatment of cancer encompasses the treatment, imaging, diagnosis, prevention, inhibition and reduction of tumor growth.

[0235] This aspect of the invention may also be expressed as a method for targeting PTK7 expressing cells, particularly PTK7 expressing tumor cells, comprising contacting an antibody conjugate according to the invention with cells that may express PTK7. Thus, the method according to this aspect is suitable for determining whether a cell is PTK7 expressing. 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 may express PTK7 are PTK7 expressing cells. Targeting PTK7 expressing cells preferably includes one or more of treating, imaging, diagnosing, preventing, inhibiting, and reducing the growth of PTK7 expressing cells, particularly PTK7 expressing tumor cells. The method according to this embodiment may be medical or non-medical. Non-medical methods according to this aspect may involve in vitro or ex vivo targeting of PTK7 expressing cells, where the cells that may express PTK7 are present, for example, in a sample taken from a patient. Such non-medical methods are typically used for the diagnosis of cancer, particularly PTK7 positive cancer.

[0236] In the context of the present invention, the subject may suffer from a disorder selected from 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.

[0237] The inventors have surprisingly found that the antibody conjugates according to the 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 invention is increased compared to conventional PTK7-targeting antibody conjugates.

[0238] Conjugation Style In the context of the present invention, "conjugation mode" refers to the process used to conjugate the payload D to the antibody AB, as well as the structural features of the resulting antibody conjugate, in particular the linker connecting the payload to the antibody, that are a direct result of the conjugation process. Thus, in one embodiment, the conjugation mode refers to the process for conjugating the payload to the antibody. In an alternative embodiment, the conjugation mode refers to the structural features of the linker and / or the attachment point of the linker to the antibody that are a direct result of the process for conjugating the payload to the antibody.

[0239] 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, the conjugation mode being used to connect an antibody AB with a payload D via a linker L. The conjugation mode comprises: (i) reacting an antibody containing y core N-acetylglucosamine (GlcNAc) moiety (where y=1, 2, 3, or 4) with an antibody of formula S(F) in the presence of a catalyst x -P(in the formula, S(F) x is a sugar derivative containing x reactive groups F capable of reacting with a reactive group Q, where x is 1 or 2, P is a nucleoside mono- or diphosphate, and the catalyst is S(F) x (which can transfer a moiety to the core-GlcNAc moiety) AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) (In the formula, -AB is an antibody capable of targeting PTK7-expressing tumors, -Fuc is fucose, - obtaining a modified antibody according to (ii) coupling the modified antibody to a compound represented by the structure (2): QLD (2) (In the formula, -Q is a reactive moiety, - L is a linker connecting Z to D, -D is reacted with a compound according to the formula (I) selected from the group consisting of anthracyclines, camptothecins, tubulysins, enediynes, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, BCL-XL inhibitors, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and analogs or prodrugs thereof; and obtaining an antibody conjugate according to structure (1).

[0240] Preferably, increasing the therapeutic index of the antibody conjugate comprises: (a) increasing the therapeutic efficacy of the antibody conjugate, and / or (b) is selected because it increases the tolerability of the antibody conjugate.

[0241] The increased therapeutic efficacy of the antibody conjugate according to the invention may take the form of a reduced tumor size and / or a prolonged period of regression when compared to conventional PTK7-targeted ADCs. The increased tolerability of the antibody conjugate according to the invention may take the form of a reduced sign of toxicity when compared to administration of a PTK7-targeted ADC made by conventional techniques. The reduced symptoms may also be referred to as a reduction in symptoms or side effects of cancer treatment and may involve one or more clinical signs, such as reduced weight loss, reduced mobility reduction, reduced food intake reduction, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology. EXAMPLES

[0242] General procedure for transient expression and purification of monoclonal antibodies: Various IgGs (cofetuzumab, 4D5, 7C8, or B12) were transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 1 L, 100 mL, 100 mL, and 5 L scales, respectively. Other IgGs (12C6 and 12C6a) were transiently expressed in ExpiCHO-S™ cells (Gibco) at 500 mL scale. Non-fucosylated 12C6 was transiently expressed in CHO cells using ProBioGen's GlymaxX® technology by Evitria. Supernatants were purified using HiTrap MabSelect sure columns. The supernatant was loaded onto the column and then washed with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl (TBS). The retained protein was eluted with 0.1 M AcOH (pH 2.7). The eluted product was immediately neutralized with 2.5 M Tris-HCl pH 8.8 and dialyzed against 20 mM histidine, 150 mM NaCl, pH 7.5. The IgG was then concentrated (>20 mg / mL) using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). The sequence of the IgG is shown below:

[0243] Cofetuzumab (I) light chain (SEQ ID NO: 8):

number

[0244] Cofetuzumab (I) heavy chain (SEQ ID NO: 7):

number

[0245] 4D5(II) light chain (SEQ ID NO: 16):

number

[0246] 4D5(II) Heavy Chain (SEQ ID NO: 15):

number

[0247] 7C8(III) Light Chain (SEQ ID NO:25):

number

[0248] 7C8(III) Heavy Chain (SEQ ID NO:26):

number

[0249] 12C6(IV) light chain (SEQ ID NO:20):

number

[0250] 12C6(IV) Heavy Chain (SEQ ID NO: 19):

number

[0251] 12C6a(V) light chain (SEQ ID NO:22):

number

[0252] 12C6a(V) heavy chain: See 12C6 heavy chain (SEQ ID NO: 19).

[0253] B12(VI) light chain (SEQ ID NO:28):

number

[0254] B12(VI) Heavy Chain (SEQ ID NO:27):

number

[0255] General procedure for analytical RP-UPLC (DTT-treated samples): Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 μL) and incubated at 37° C. for 15 min. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD. Samples (5 μL) were eluted on a Bioresolve RP mAb2.1 column at a column temperature of 70° C. * Injections were performed at 0.4 mL / min into a 150 mm 2.7 μm column (Waters). A linear gradient was applied from 30 to 54% acetonitrile in 0.1% TFA and water in 9 min. The absorbance of the eluted peaks was measured at 215 nm followed by automated integration (MassLynx, Waters) to determine reaction conversion.

[0256] General procedure for mass spectrometry analysis of (modified) monoclonal antibodies: Prior to mass spectrometry, IgG was treated with IdeS, which allows the analysis of Fc / 2 fragments. For the analysis of both light and heavy chains, a solution of 20 μg of (modified) IgG was incubated for 5 min at 37° C. with 100 mM DTT in a total volume of 4 μL. Azide functions, if present, are reduced to amines under these conditions. For the analysis of Fc / 2 fragments, a solution of 20 μg of (modified) IgG was incubated for 1 h at 37° C. with IdeS / Fabricator™ (1.25 U / μL) in phosphate buffered saline (PBS) pH 6.6 in a total volume of 10 μL. After dilution of the samples to 80 μL, electrospray ionization time of flight (ESI-TOF) was analyzed on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0257] General procedure 2 for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc): IgG (15 mg / mL) was incubated with 1% w / w EndoSH (see Examples 1-3 and SEQ ID NO: 1 as described in PCT / EP2017 / 052792, which is incorporated herein by reference), 3% w / w His-TnGalNAcT (see Examples 3 and 4 and SEQ ID NO: 49 as described in PCT / EP2016 / 059194, which is incorporated herein by reference), 6 mM MnCl2 and 0.01% AP (Roche) and UDP6-N3-GalNAc (compound 2d in FIG. 3, 25 equivalents compared to IgG) in TBS for 16 hours at 30° C. The functionalized IgG was then purified using a HiTrap MabSelect sure 5 mL column. After loading the reaction mixture, the column was washed with TBS+0.2% Triton and TBS. IgG was eluted with 0.1 M AcOH (pH 2.7) and neutralized with 2.5 M Tris-HCl pH 8.8. After three dialysis into 20 mM histidine, 150 mM NaCl pH 7.5, IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0258] Preparation of azide-functionalized antibodies: Examples 1-7: Example 1: Preparation of Cofetuzumab-(6-N3-GalNAc)2(I-N3) Cofetuzumab was converted to cofetuzumab-(6-N3-GalNAc)2 following a general procedure for enzymatic remodeling. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 24329 Da, ∼90% of total Fc / 2) corresponding to the expected product.

[0259] Example 2: Preparation of B12-(6-N3-GalNAc)2 (VI-N3) Following the general procedure for enzymatic remodeling, B12 was converted to B12-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 24330 Da, approx. 90% of total Fc / 2) corresponding to the expected product.

[0260] Example 3: Preparation of 4D5-(6-N3-GalNAc)2(II-N3) Following the general procedure for enzymatic remodeling, 4D5 was converted to 4D5-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, ∼90% of total Fc / 2) and one minor Fc / 2 product corresponding to the non-fucosylated product (observed mass 24186 Da, ∼10% of total Fc / 2).

[0261] Example 4: Preparation of 7C8-(6-N3-GalNAc)2(III-N3) Following the general procedure for enzymatic remodeling, 7C8 was converted to 7C8-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, ∼90% of total Fc / 2) and one minor Fc / 2 product corresponding to the non-fucosylated product (observed mass 24186 Da, ∼10% of total Fc / 2).

[0262] Example 5: Preparation of 12C6-(6-N3-GalNAc)2 (IV-N3) Following the general procedure for enzymatic remodeling, 12C6 was converted to 12C6-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the expected product (observed mass 24331 Da, ∼90% of total Fc / 2) and one minor Fc / 2 product corresponding to the non-fucosylated product (observed mass 24186 Da, ∼10% of total Fc / 2).

[0263] Example 6: Preparation of 12C6a-(6-N3-GalNAc)2(V-N3) Following the general procedure for enzymatic remodeling, 12C6a was converted to 12C6a-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the expected product (observed mass 24332 Da, ∼90% of total Fc / 2) and one minor Fc / 2 product corresponding to the non-fucosylated product (observed mass 24186 Da, ∼10% of total Fc / 2).

[0264] Example 7: Preparation of aFuc-12C6-(6-N3-GalNAc)2 (aFuc-IV-N3) Following the general procedure for enzymatic remodeling, aFuc-12C6 was converted to aFuc-12C6-(6-N3-GalNAc)2. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 24187 Da, ∼95% of total Fc / 2) corresponding to the expected product.

[0265] Examples 8-12: Synthesis of linker conjugates 3, 4, 5b, and 9 [ka] Example 8: Preparation of Compound 11 Compound 10 (163 mg, 240 μmol) was added to a mixture of exatecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMF (0.9 mL). After 20 h, the reaction mixture was diluted with 9 mL of DCM and purified by gradient column chromatography (0→40% MeOH / DCM) to give 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 FN6O 10 + (M+H) + Calculated 977.39, found 977.72. In addition to 11, exatecan free base (82.4 mg, 189 μmol, 20%) was recovered. 24 H 23 FN3O4 + (M+H) + The calculated value was 436.46 and the actual value was 436.54.

[0266] Example 9. Preparation of Compound 3 The synthesis of BCN-HS-(va-PABC-Ex)2 (3) is also described in PCT / EP2021 / 075401 (Example 4), which is incorporated herein. To a solution of compound 11 (155 mg, 159 μmol) in DMF (1.6 mL) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 12 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL) and purified by gradient column chromatography (0→40% MeOH / DCM) to give 3 as a pale yellow solid (94 mg, 44 μmol, 28%). LCMS (ESI+)C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + The calculated value was 1066.88, and the actual value was 1067.12. [ka]

[0267] Example 10: Preparation of linker conjugate 4 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 triethylamine (22 μL, 16 mg, 158 μmol) and a solution of vc-PABC-MMAE.TFA13 (96 mg, 78 μmol) in DMF (1.0 mL). The mixture was allowed to stand for 19 h and 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) in water (1% AcOH)). The desired product 4 was obtained as a colorless film (41 mg, 14.7 μmol, 45%). + ) C 138 H 219 N 23 O 35 S 2+ (M+2H + ) calculated value 1395.79, actual value 1396.31.

[0268] Example 11: Synthesis of linker conjugate 5b The synthesis of BCN-HS-vc-PABC-CM (5b) was prepared according to the procedure described in WO2019110725, which is incorporated herein. To a solution of 15 (9.0 mg, 4.88 μmol, compound 138 in WO2019110725) in DMF (445 μL) was added a solution of 16 (12.8 mg, 24.4 μmol, compound 3 in WO2019110725) in DMF (45.8 μL) and a solution of 50% v / v Et3N in DMF (8.98 μL, 32.2 μmol). The mixture was allowed to stand for 4 h and then diluted with DCM to a total volume of 4.5 mL. The mixture was purified by silica gel chromatography (0→20% MeOH in DCM). The desired product 5b was obtained as a colorless oil (5.0 mg, 2.24 μmol, 46%). LCMS(ESI+)C 96 H136 IN9O 35 S4 2+ (M+2H + ) calculated value 1115.35, actual value 1115.40. [ka]

[0269] Example 12. Preparation of Compound 9 A solution of BCN-HS-PEG2-b-(Glu(OFm)-OH)2 (8, 12.1 mg, 10 μmol, 1.0 equiv.) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-exatecan (5b, Fmoc-deprotected 5, 19 mg, 25 μmol, 2.5 equiv.) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 equiv.) and HATU (8.9 mg, 23 μmol, 2.3 equiv.). After stirring at room temperature for 2 h, the reaction mixture was further diluted with DCM (800 μL) and purified by flash column chromatography on silica gel (0%→20% MeOH in DCM) to give the product as a clear oil (the presence of DMF makes it difficult to determine the yield). LCMS (ESI+)C 140 H 150 F2N 17 O 33 S + (M / 2+H + ) calculated value 1334.01, actual value 1334.79.

[0270] This compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 equiv.) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by flash column chromatography over silica gel (0%→45% MeOH in DCM) to give compound 9 as a yellow solid in 44% yield (10.2 mg, 4.4 μL). LCMS (ESI+)C 112 H 130 F2N 17 O 33 S + (M / 2+H +) calculated value 1156.2, actual value 1156.74.

[0271] Examples 13-24: Conjugation of linker payload to (modified) monoclonal antibodies Example 13: Preparation of Cofetuzumab-Aur0101 Cofetuzumab (14.4 mg, 4.34 mg / ml in 20 mM histidine, 6% sucrose) was charged 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 for 2 hours at room temperature. The reduced antibody was charged with DMA to give 10% v / v and 6 molar equivalents of auristatin 0101 (2 mM in DMA) and incubated for 1 hour at room temperature. The reaction was quenched using 6 molar equivalents of NAC (100 mM in water). The conjugate was purified by preparative discontinuous diafiltration on a Vivaspin into 20 mM histidine / 6% sucrose. 0.04% Tween-20 was added before filter sterilization. The average DAR was measured to be 4.3.

[0272] Example 14: Conjugation of cofetuzumab (6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate cofetuzumab-3 To a solution of cofetuzumab (6-N3-GalNAc)2 (1664 μL, 32.0 mg, 19.37 mg / ml in TBS pH 7.5) was added sodium deoxycholate (322 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (86 μL, 10 mM solution in DMF) and propylene glycol (881 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified on a HiLoad 16 / 600 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). To remove excess free payload, 25 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (0.20 μm pores, Corning). The solution was subsequently buffer exchanged using a HiTrap26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26461 Da, approximately 90% of total Fc / 2) corresponding to the conjugate cofetuzumab-3. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.86.

[0273] Example 15: Conjugation of cofetuzumab (6-N3-GalNAc)2 with BCN-HS-(vc-PABC-MMAE)24 to obtain conjugate cofetuzumab-4. To a solution of cofetuzumab (6-N3-GalNAc)2 (463 μL, 9.0 mg, 19.37 mg / ml in TBS pH 7.5), sodium deoxycholate (60 μL, 110 mM) and BCN-HS-(vc-PABC-MMAE)24 (1.8 μL, 100 mM solution in DMF) and DMF (58 μL) were added. The reaction was incubated overnight at room temperature. The conjugate was then 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 equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 27119 Da, approximately 90% of total Fc / 2) corresponding to the conjugate cofetuzumab-4. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.90.

[0274] Example 16: Conjugation of cofetuzumab (6-N3-GalNAc)2 with BCN-HS-vc-PABC-calicheamicin 5b to obtain conjugate cofetuzumab-5b. To a solution of cofetuzumab (6-N3-GalNAc)2 (730 μL, 14.0 mg, 19.37 mg / ml in TBS pH 7.5) was added sodium deoxycholate (94 μL, 110 mM) and BCN-HS-vc-PABC-calicheamicin 5b (7 μL, 40 mM solution in DMF) and DMF (87 μL). The reaction was incubated overnight at room temperature. The conjugate was then 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 equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the samples after IdeS treatment showed one major Fc / 2 product (observed mass 26714 Da, approximately 90% of total Fc / 2) corresponding to the conjugate cofetuzumab-5b. RP-UPLC analysis of the samples under reducing conditions showed an average DAR of 1.93.

[0275] Example 17: Conjugation of B12(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain cofetuzumab B12-3 To a solution of B12(6-N3-GalNAc)2 (6.33 mL, 150.0 mg, 23.71 mg / ml in TBS pH 7.5) was added BCN-HS-PEG2-HS-(va-PAB-Ex)23 (495 μL, 10 mM solution in DMF) and propylene glycol (7.0 mL). The reaction was incubated overnight at room temperature. The conjugate was then purified on a HiLoad 26 / 600 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). To remove excess free payload, 150 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (0.20 μm pore size, Corning). The solution was then dialyzed against 20 mM histidine, 6% sucrose buffer pH 6.0 for 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 the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26462 Da, approximately 90% of total Fc / 2) corresponding to the conjugate B12-3. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.75.

[0276] Example 18: Conjugation of 4D5(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 4D5-3 To a solution of 4D5(6-N3-GalNAc)2 (424 μL, 8.8 mg, 20.74 mg / ml in TBS pH 7.5) was added sodium deoxycholate (88 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (23.4 μL, 10 mM solution in DMF) and propylene glycol (240 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). To remove excess free payload, 12 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (0.20 μm pore size, Corning). The solution was then dialyzed three times and supplemented with 20 mM histidine, 6% sucrose buffer (pH 6.0), and 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26465 Da, approx. 90% of total Fc / 2) corresponding to the conjugate 4D5-3, and one minor Fc / 2 product (observed mass 26318 Da, approx. 10% of total Fc / 2) corresponding to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.91.

[0277] Example 19: Conjugation of 7C8(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 7C8-3 To a solution of 7C8(6-N3-GalNAc)2 (313 μL, 6.9 mg, 22.14 mg / ml in TBS pH 7.5) was added BCN-HS-PEG2-HS-(va-PAB-Ex)23 (23.1 μL, 10 mM solution in DMF) and propylene glycol (254 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified on a Superdex200 10 / 300 enrichment column (Cytiva) on an AKTA Pure (Cytiva). To remove excess free payload, 11 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (0.20 μm pore size, 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 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26466 Da, approximately 90% of total Fc / 2) corresponding to the conjugate 7C8-3. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.95.

[0278] Example 20: Conjugation of 12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 12C6-3 To a solution of 12C6(6-N3-GalNAc)2 (481 μL, 10.1 mg, 21.01 mg / ml in TBS pH 7.5) was added BCN-HS-PEG2-HS-(va-PAB-Ex)23 (33.7 μL, 10 mM solution in DMF) and propylene glycol (370 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). To remove excess free payload, 12 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (0.20 μm pore size, Corning). The solution was subsequently buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH, equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, and then filter-sterilized. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26466 Da, approximately 95% of total Fc / 2) corresponding to the conjugate 12C6-3, and one minor Fc / 2 product (observed mass 26318 Da, approximately 5% of total Fc / 2) corresponding to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.85.

[0279] Example 21: Conjugation of 12C6a(6-N3-GalNAc)2 with BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 12C6a-3 To a solution of 12C6a(6-N3-GalNAc)2 (484 μL, 10.9 mg, 22.53 mg / ml in TBS pH 7.5) was added BCN-HS-PEG2-HS-(va-PAB-Ex)23 (36.3 μL, 10 mM solution in DMF) and propylene glycol (400 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified on a Superdex200 10 / 300 enrichment column (Cytiva) on an AKTA Pure (Cytiva). To remove excess free payload, 12 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation followed by filtration through a PES syringe filter (0.20 μm pore size, Corning). The solution was then dialyzed three times and supplemented with 20 mM histidine, 6% sucrose buffer (pH 6.0), and 0.04% Tween-20 before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26466 Da, approximately 95% of total Fc / 2) corresponding to the conjugate 12C6a-3. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.90.

[0280] Example 22: Conjugation of 12C6(6-N3-GalNAc)2 with BCN-HS-PEG2-(eva-PAB-Ex)29 to obtain conjugate 12C6-9 To a solution of 12C6(6-N3-GalNAc)2 (340 μL, 10 mg, 29.45 mg / ml in TBS pH 7.5) was added sodium deoxycholate (100 μL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)29 (20 μL, 10 mM solution in DMF) and propylene glycol (280 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). To remove excess free payload, 12 mg of activated charcoal (Carbon RHC, Filtrox AG) was added and rotated overnight. The charcoal was removed by centrifugation and subsequently filtered through a PES syringe filter (0.20 μm pore size, Corning). The solution was then buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20, and then filter-sterilized. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26466 Da, approx. 95% of total Fc / 2) corresponding to the conjugate 12C6-9, and one minor Fc / 2 product (observed mass 26643 Da, approx. 5% of total Fc / 2) corresponding to the non-fucosylated product. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.84.

[0281] 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 To a solution of aFuc-12C6(6-N3-GalNAc)2 (549 μL, 13 mg, 23.69 mg / ml in TBS pH 7.5) was added sodium deoxycholate (130 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (34.7 μL, 10 mM solution in DMF) and propylene glycol (355 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). The solution was subsequently 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, and 0.04% Tween-20, before filter sterilization. Mass spectrometry analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 26318 Da, approximately 95% of total Fc / 2) corresponding to the conjugate aFuc-12C6-3. RP-UPLC analysis of the sample under reducing conditions showed an average DAR of 3.74.

[0282] 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 To a solution of aFuc-12C6(6-N3-GalNAc)2 (59 μL, 1.4 mg, 23.69 mg / ml in TBS pH 7.5) was added sodium deoxycholate (14 μL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)29 (2.8 μL, 10 mM solution in DMF) and propylene glycol (39 μL). The reaction was incubated overnight at room temperature. The conjugate was then purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). RP-UPLC analysis of the conjugated aFuc-12C6-9 sample under reducing conditions showed an average DAR of 3.81.

[0283] Examples 25-28: In vitro assays Example 25. hPTK7, cPTK7 and rPTK7 Binding Assays to mAbs and ADCs Using ELISA Nickel NTA plates (Pierce™ Nickel-Coated Plates, ThermoScientific™) were washed three times before use. Human PTK7 (CCK4 Protein, ECD, His Tag, Sino Biological), Cynomolgus PTK7 (CCK4 Protein, ECD, His Tag, Sino Biological), and Rat PTK7 (His Tag, Acro Biosystems) were dissolved at a concentration of 0.05 μg / mL in 0.1% BSA in PBS (PBA). 100 μL was added to each well and incubated at room temperature for 1 hour with shaking. After removal, the plates were washed three times with 0.05% Tween-20 in PBS (wash buffer). ADC was diluted in 0.1% PBA to a final concentration of 8 μg / mL and 100 μL was added to each well (in triplicate). ADC was incubated at room temperature for 1 hour. Plates were washed three times with wash buffer before adding 100 μL of a 1:1000 dilution of secondary antibody (goat anti-human IgG, HRP conjugate, Invitrogen). Plates were again incubated for 1 hour at room temperature and then washed three times with wash buffer and three times with PBS. Finally, 100 μL of TMB ELISA substrate (1 Step™ Turbo TMB ELISA Substrate, ThermoScientific™) was added and incubated for 1 minute. Absorbance of the colorimetric signal was measured at 652 nm on an Infinite® M1000 (Tecan). Data were plotted corrected for background (see Figures 8A, 8B and 11).

[0284] Example 26. Human and mouse serum stability The stability of the ADC in mouse and human plasma was tested. Prior to the assay, plasma was depleted of all IgG using CaptivA® Protein A agarose (1 mL agarose / mL serum). ADC was added to the depleted human / mouse serum to a final concentration of 0.1 mg / mL followed by incubation at 37°C. At each time point, 0.5 mL was flash frozen and stored at -80°C until further analysis. To isolate the ADC after incubation, 20 μl of CaptivA® Protein A agarose resin was added to the sample and incubated for 1 h at room temperature. The resin was washed three times with PBS followed by the addition of 0.1 M glycine-HCl (pH 2.7) (0.4 mL) to elute the ADC. After elution, the sample was immediately neutralized with 1.0 M Tris pH 8.0 (0.1 mL). Samples were spin filtered three times against PBS using Amicon Ultra spin filters 0.5 mL MWCO 10 kDa (Merck Millipore) to reduce the volume to 40 μL, giving a final ADC concentration of approximately 1 mg / mL. Samples were analyzed by RP-UPLC (DTT reduced) to determine the DAR (table below). [Table 1] [Table 2] -†Signal too low *Direct measurement (not by separation from plasma with protA agarose resin). The numbers in brackets are the percentages of t=0.

[0285] Example 27: In vitro cytotoxicity 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. ADCs were added in triplicates at the square root of 10 dilution series to give final concentrations ranging from 100 nM to 0.01 nM. Cells were incubated for 5 days at 37 °C and 5% CO2 in a humidified atmosphere. Culture medium was replaced with resazurin (Sigma Aldrich) at 0.01 mg / mL in DMEM supplemented with 10% FBS (200 μL / well). After approximately 4 hours in a humidified atmosphere at 37°C and 5% CO2, fluorescence was detected using a fluorescence plate reader (Infinite® M1000 Tecan) at excitation of 560 nm and emission of 590 nm. Relative fluorescence units (RFU) were normalized to cell viability percentage by setting wells without cells as 0% viability and wells with untreated cells as 100% viability (see Figure 9). IC of ADC on A431 cells 50 Values ​​were calculated by non-linear regression using Graphpad prism software and are shown in the table below. [Table 3]

[0286] Example 28. Binding affinity of 12C6-3 and 12C6 naked mAbs with FcγR using Biacore Binding of test antibodies to high affinity and low affinity human Fc gamma receptors was assessed by single cycle analysis using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software (Cytiva, Uppsala, Sweden) running at a flow rate of 30 μl / min. Human Fc receptors, FcγRI, FcγRIIA (both 167R and 167H polymorphisms), FcγRIIB, FcγRIIIA (both 176F and 176V polymorphisms), and FcγRIIIB were obtained from Sino Biological (Beijing, China). FcγRs were captured using standard amine chemistry onto a CM5 sensor chip pre-coupled using a His capture kit (Cytiva, Uppsala, Sweden).

[0287] At the start of each cycle, His-tagged Fc gamma receptor (ligand) diluted in HBS-P+ (Cytiva, Uppsala, Sweden) was loaded to a specific RU level. A 5-point, 3-fold dilution range of test antibody (analyte) with no regeneration between each concentration was used for each receptor tested. Test antibody was passed over the chip in increasing concentrations at 30 μl / min, followed by a single dissociation step. After dissociation, the chip was regenerated by injection of glycine pH 1.5. The signal from the reference channel Fc1 (blank) was subtracted from the signal of the receptor-loaded Fc to correct for differences in nonspecific binding to the reference surface. Sensorgrams were analyzed by 1:1 kinetics of the high affinity Fc gamma receptor hFcγRI and steady-state binding of the low affinity Fc gamma receptor.

[0288] Table 4. Summary table of binding of test antibodies and ADCs to different human Fcγ receptors. The relative scale of binding (KD) is +++++, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 21 -9 ~10 -10 M range,++++,10 -8 M range, +++, 10 -7 Range of M,++,10 -6 M, +, 10 -5 The range of M is shown. [Table 4-1]

[0289] Examples 29-34: CDX evaluation and in vivo studies Example 29: IHC staining of NCI-H446 for PTK7 (performed at Crown Bioscience Taicang, China) Formalin-fixed paraffin-embedded (FFPE) blocks were cut onto glass slides at a thickness of 4 μm on a manual rotary microtome. Slides were baked at 60 °C for 30 min. A Bond RX autostainer (Leica) was used to stain as follows: slides were first treated with dewaxing solution (Leica) for 0.5 min at 72 °C, then at room temperature, transferred to alcohol and treated three times at room temperature. Slides were then washed three 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. Slides were then washed four times briefly in Bond wash buffer, followed by a 3-min wash at room temperature. A peroxide block was performed at room temperature for 10 min, followed by three washes again. The primary antibody rabbit anti-human PTK7 (PA5-82070, Thermo Fisher, 0.25 μg / mL) was then added and incubated at room temperature for 1 h. This was followed by one quick wash and three washes of 2 min in Bond wash buffer. Secondary antibody goat anti-rabbit HRP (Leica, ready to use) was added and incubated for 20 min at room temperature, followed by one quick wash and three washes of 2 min in Bond wash buffer. Slides were rinsed with deionized water and then incubated with 3,3'-diaminobenzidine (DAB) Refine for 5 min at room temperature. After three washes with deionized water, slides were treated with hematoxylin as a contrast dye for 10 min. Finally, all slides were washed with deionized water and Bond wash buffer.

[0290] All stained sections were scanned at 40x magnification with a NanoZoomer-HT 2.0 imaging system. All images of non-necrotic tumor areas were analyzed with the HALO™ platform. The intensity of membrane positive staining was scored at four levels: 0 (negative), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). The percentage of cells at different intensity levels was evaluated with an H-score: H-score=(% at 0)×0+(% at 1)×1+(% at 2)×2+(% at 3)×3 (H-score range is 0-300). See Table 4 for H-scores of NCI-H446 lung cancer xenograft cell line. [Table 4-2]

[0291] Example 30: In vivo efficacy study (performed at Crown Bioscience, Zhongshan, China) NCI-H446, a lung cancer xenograft model cell line, was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with standard 5% CO2 specifications. Exponentially growing cells were harvested and counted for tumor inoculation.

[0292] BALB / c nude mice (female) aged 7–9 weeks were treated with 5 × 10 cells in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 6 Tumor cells were injected subcutaneously into the right anterior flank region. 3 Once the average size of the mice reached, they were randomized into 10 groups of 8 mice each and treatment was initiated. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0293] Electronic caliper measurements were taken twice weekly. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice weekly. The experimental endpoints were 3,000 mm 3or greater than 20% tumor volume, or weight loss over 28 days, whichever occurs first.

[0294] Randomized: Mean tumor size approximately 100-200 mm 3 Randomization was performed once the 100% CI of mice was reached. A total of 80 mice were enrolled in the NCI-H446 model study and randomly assigned to 10 groups with 8 mice per group. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0295] Administration of test articles: Test articles were administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment began on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0296] Observations and Data Collection. After tumor cell inoculation, animals are checked daily for morbidity and mortality. At the time of routine monitoring, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using calipers, and volumetric data are expressed in mm3 using the formula V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. [Table 5]

[0297] The tumor volume and mouse body weight over time in the efficacy test using the above test items are shown in Figures 10A to 10D.

[0298] Example 31: In vivo efficacy studies NCI-H446, a lung cancer xenograft model cell line, was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with standard 5% CO2 specifications. Exponentially growing cells were harvested and counted for tumor inoculation.

[0299] BALB / c nude mice (female) aged 7–9 weeks were treated with 5 × 10 cells in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 6 Tumor cells were injected subcutaneously into the right anterior flank region. 3 Once the average size of the mice reached, they were randomized into 6 groups of 8 mice and treatment was initiated. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0300] Electronic caliper measurements were taken twice weekly. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice weekly. The experimental endpoints were 3,000 mm 3 tumor volume of 0.01 mg / kg or greater, or weight loss over 77 days, whichever occurs first.

[0301] Randomized: Mean tumor size approximately 136 mm 3 Randomization was performed once the 0.01% CI of mice was reached. A total of 48 mice were enrolled in the NCI-H446 model study and randomly assigned to six groups with 8 mice per group. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0302] Administration of test articles: Test articles were administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment began on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0303] Observations and data collection: After tumor cell inoculation, animals are checked daily for morbidity and mortality. At the time of routine monitoring, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using calipers, and volumetric data are expressed in mm3 using the formula V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. [Table 6]

[0304] Tumor volumes and mouse body weights over time in the efficacy study and corresponding Kaplan-Meier plots using the test parameters listed above are shown in Figures 12A, 12B, and 12C.

[0305] Example 32: In vivo efficacy studies BALB / c nude mice (female) aged 7–9 weeks were loaded with tumor fragments of BR1282, a xenograft model cell line derived from a breast cancer patient. Tumor fragments (2–3 mm in diameter) were inoculated subcutaneously in the right upper flank region. Tumors were 100–200 mm 3 Once the average size of the mice reached, they were randomized into 9 groups of 8 mice each and treatment was initiated. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0306] Electronic caliper measurements were taken twice weekly. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice weekly. The experimental endpoints were 3,000 mm 3 tumor volume, greater than 20%, or weight loss over 39 days, whichever occurs first.

[0307] Randomized: Mean tumor size ≈135 mm 3 Randomization was performed once the BR1282 model study was completed. A total of 72 mice were enrolled in the BR1282 model study and randomly assigned to 9 groups with 8 mice per group. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0308] Administration of test articles: Test articles were administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment began on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0309] Observations and data collection: After tumor cell inoculation, animals are checked daily for morbidity and mortality. At the time of routine monitoring, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using calipers, and volumetric data are expressed in mm3 using the formula V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. [Table 7]

[0310] Tumor volume and mouse body weight over time in efficacy studies using the above test items are shown in Figures 13A and 13B.

[0311] Example 33: In vivo efficacy studies NCI-H446, a lung cancer xenograft model cell line, was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in a humidified cell culture incubator at 37°C with standard 5% CO2 specifications. Exponentially growing cells were harvested and counted for tumor inoculation.

[0312] BALB / c nude mice (female) aged 7–9 weeks were treated with 5 × 10 cells in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 6 Tumor cells were injected subcutaneously into the right anterior flank region. 3 Once the average size of the mice reached, they were randomized into 5 groups of 8 mice and treatment was initiated. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0313] Electronic caliper measurements were taken twice weekly. Clinical signs, food and water consumption, and behavioral changes were observed daily, and animals were weighed twice weekly. The experimental endpoints were 3,000 mm 3 or greater than 20% tumor volume, or weight loss over 28 days, whichever occurs first.

[0314] Randomized: Mean tumor size approximately 100-200 mm 3 Randomization was performed once the 0.01% CI was reached. A total of 40 mice were enrolled in the NCI-H446 model study and randomly assigned to 5 groups with 8 mice per group. Randomization was performed based on the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is designated as day 0.

[0315] Test Article Administration: ADC test article administration was via intravenous injection via the tail vein, with a dose of 10 mL / kg. ADC single doses only were initiated on the same day as randomization. Olaparib administration was given orally at a dose of 50 mg / kg QD x 21 days, also initiated on the same day as randomization. Dosing was performed in a laminar flow cabinet.

[0316] Observations and Data Collection: After tumor cell inoculation, animals are checked daily for morbidity and mortality. At the time of routine monitoring, animals are checked for tumor growth and adverse effects of treatment on normal behavior such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / fur matting, and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using calipers, and volumetric data are expressed in mm using the formula V = (L x W x W) / 2. 3 where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Dosing, as well as tumor and body weight measurements, are performed in a laminar flow cabinet. [Table 8]

[0317] Tumor volume and mouse body weight over time in efficacy studies using the above test items are shown in Figures 14A and 14B.

[0318] Example 34: In vivo tolerability study Six to seven week old Sprague Dawley rats (female) were allowed to acclimate for one week before enrollment in the study.

[0319] Randomization: In the tolerability study, a total of 15 rats were enrolled and randomly assigned to five study groups, with 3 rats per group. Randomization was performed based on the "matched distribution" method (StudyDirector™ software, version 3.1.399.19). The date of randomization was designated as day 0.

[0320] Test article administration: Treatment began on the same day as randomization (day 0) per study design. Dose levels selected were 75, 100, 125, and 150 mg / kg in a single dose. All animals were dosed by slow intravenous injection (10 mL / kg).

[0321] Observations and data collection: Animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for the effect of treatment on behavior such as mobility, food and water consumption, weight gain / loss, eye / hair matting, and any other abnormalities. Mortality of individual animals and clinical signs observed were recorded in detail. Dosing and weight measurements were performed in a laminar flow cabinet. Body weights were measured daily using StudyDirector™ software (version 3.1.399.19).

[0322] The rat body weight over time for 12C6-3 as a test item at several dose levels is shown in FIG.

Claims

1. Antibody conjugates conforming to general structure (1) or its pharmaceutically acceptable salts: AB-[(L ６ ) ｂ -{Z-L-D} ｘ ] ｙ （１） During the ceremony, AB comprises a light chain and a heavy chain, and the light chain and the heavy chain are anti-PTK7 antibodies containing the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 19, respectively. L ６ is -GlcNAc(Fuc) ｗ -(G) ｊ -S-(L ７ ) ｗ’ - and During the ceremony, G is selected from galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, or N-acetylneuraminic acid. j is an integer in the range of 0 to 10. S is GalNAc, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is either 0 or 1, w' is 0, 1, or 2. L ７ is -N(H)C(O)CH ２ -, -N(H)C(O)CF ２ -, or -CH ２ - and also, Z-L-D is 【Transformation 7】 In the equation, * represents the junction point between Z-L-D and L6. b is either 0 or 1, x is either 1 or 2, y is 1, 2, 3, or 4.

2. The antibody conjugate according to claim 1, wherein j is 0.

3. The antibody conjugate according to claim 1, wherein w' is 0.

4. The antibody conjugate according to claim 1, wherein x is 1.

5. The antibody conjugate according to claim 1, wherein y is 2.

6. The antibody conjugate according to claim 1, wherein the antibody conjugate is the following formula or a pharmaceutically acceptable salt thereof: 【Transformation 8】

7. The antibody conjugate according to claim 1, wherein the antibody conjugate is the following formula or a pharmaceutically acceptable salt thereof: 【Chemistry 9】

8. The antibody conjugate according to claim 1, wherein w is 0.

9. The antibody conjugate according to claim 1, wherein w is 1.

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