Antibody conjugates for targeting tumors expressing PTK7

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] This invention relates to the field of bioconjugation. More specifically, it relates to antibody-drug conjugates for targeted therapy of patients with cancer, particularly PTK7-expressing tumors. [Background technology]

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

[0003] Current ADCs are generally prepared by various conjugation techniques (summarized in Figure 1), primarily based on conjugation to cysteine ​​side chains with maleimide or lysine side chains with activated esters. To produce ADCs based on native cysteine ​​naturally involved in the disulfide bond, thiols in the side chain can be released by exposing the antibody to a suitable reducing agent such as TCEP or DTT, followed by treatment with a maleimide-functionalized linker drug. The resulting ADC typically consists of a mixture of positional isomers, where only the sum of eight free thiols is not comprehensively alkylated. Alternatively, to produce site-specific ADCs, antibodies can be generated by mutating one or more amino acids to cysteine ​​at defined positions in the sequence, and their side chains can be selectively released for alkylation by the reduction-oxidation sequence. Commonly used cysteines for site-specific conjugation are LC-41C (light chain 41C), HC-41C (heavy chain 41C), LC-80C, HC-118C, HC-265C, HC-140C, LC-149C, LC-124C, LC-180C, HC-190C, HC-160C, LC-183C, HC-290C, LC-205C, HC-220C, HC-239C, and HC-442C. Additional cysteines, 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 reactions with natural amino acid side chains, certain non-natural (non-standard) amino acids can also be manipulated within the amino acid sequence of an antibody, thereby providing unique handles for chemical conjugations such as ketones, acetylenes, azides, cyclic alkynes, or cyclic alkenes, respectively, for reactions with oximes, azides, alkynes, or tetrazines. However, the drawback of the latter approach is that, in addition to being time-consuming and expensive, it can lead to instability issues, as the natural sequence of the antibody must be redesigned.

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

[0005] Chemoenzyme conjugation via glycans, including the initial trimming of the glycan, is known in the art and has been described by van Geel et al, Bioconjugate Chem. 2015, 26, 2233, and is schematically shown in Figure 2. Specifically, a monoclonal antibody is treated with endoglycosidase to trim the glycan to core GlcNAc (directly bound to Asn-297), followed by the transfer of the azide-modified sugar under the action of glycosyltransferase. Various structures of UDP-azide sugars are shown in Figure 3. One particularly preferred combination involves the transfer of GalNAz 2b (2-azidoacetyl-N-galactosamine) under the action of mutant galactosyltransferase GalT (Y289L) disclosed in WO2007 / 095506, EP2911699B1 and van Geel et al. Another useful combination involves GlcNAz having 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 ​​for application in metal-free click chemistry are known in the art (Figure 4). In particular, various cyclooctynes ​​such as DIBO(I), DBCO / DIBAC(J), s-DIBO(K), BCN(L), and TMTHSI(T) are commonly applied for conjugation to azides.

[0007] The payload of an ADC is typically an IC in the low nanomolar or picomolar range. 50 These are highly cytotoxic molecules with a suitable value, particularly low to medium molecular weight compounds (e.g., about 200 to about 2500 Da). Examples of suitable cytotoxin classes for ADCs include anthracyclines, camptothecin, taxanes, tubulosin, enediynes, inhibitory peptides, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, hemiasterin, BCL-XL inhibitors, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs. A representative set of cytotoxic molecules and / or their synthetic derivatives or prodrugs having suitable binding sites for conjugation to monoclonal antibodies is shown in Figure 5.

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

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

[0010] Specific examples of engineers suitable for application to ADCs include, but are not limited to, calicheamycin, esperamycin, shisijimicin, and namenamemycin, as well as other engineers summarized by Galm et al., Chem. Rev. 2005, 105, 739-758.

[0011] Specific examples of auristatins suitable for application in ADCs include, but are not limited to, MMAD, MMAE, MMAF, and PF-06380101, as well as other auristatins 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, lacking observable kinase activity across various species. Genetic and biochemical studies have demonstrated the crucial function of PTK7 in non-standard Wnt signaling, and PTK7-deficient embryos exhibit severe developmental impairment in planar cell polarity. Further evidence of PTK7 function in vascular endothelial growth factor (VEGF), semaphorin / plexin, and standard Wnt signaling pathways is also available. The oncogenic function of PTK7 has been documented in colon, lung, breast, ovarian, and esophageal cancers, and PTK7 promotes cell survival and chemotherapy resistance in acute myeloid leukemia.

[0013] Antibodies against PTK7 are known in the art, as disclosed in US9777070B2 (H23 and H24, also known as 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), which are based on a humanized anti-PTK7 antibody and an auristatin microtubule inhibitor payload (Aur0101). PF-06647020 has been shown to induce prolonged tumor regression in a patient-derived tumor xenograft preclinical model 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 two or three weeks. [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] The inventors have developed an antibody conjugate that is highly suitable for targeting PTK7-expressing cells, particularly tumors. Therefore, the antibody conjugate according to the present invention is 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 a related 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. Related to this are a first medical use of the antibody conjugate according to the present invention, and a second medical use for the treatment of cancer. In a final aspect, the present invention relates to the use of a conjugation pattern to increase the therapeutic index of an antibody conjugate in the treatment of PTK7-expressing tumors.

[0017] definition As used in this description and in the claims, the verb "includes" and its conjugations are used in their non-restrictive sense, meaning that the items following the word are included, but not excluded, except for items not specifically mentioned.

[0018] In addition, the indefinite article "a" or "an" does not rule out the possibility that two or more of the elements exist, unless the context clearly requires that only one of the elements exists. Therefore, the indefinite article "a" or "an" usually means "at least one."

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

[0020] In this specification, a "hydrophilic group" or "polar linker" is defined as any molecular structure comprising one or more polar functional groups that confer improved polarity, and therefore improved water solubility, to the molecule to which it is bound. 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, acyl sulfamide groups, or carbamoyl sulfamide groups. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency, and when incorporated into antibody-drug conjugates, improved pharmacokinetics resulting in less aggregation, higher efficacy, and in vivo tolerability.

[0021] The term “salt” refers to a compound formed when an acidic proton, typically an acid proton, is replaced by a cation such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts not intended for administration to patients. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation using the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term “pharmaceutically acceptable” salt means a salt that is acceptable for administration to patients such as mammals (a salt having a counterion that has mammalian safety acceptable for a given administration plan). Such salts may be derived from a pharmaceutically acceptable inorganic or organic base and a pharmaceutically acceptable inorganic or organic acid. "Pharmacologically acceptable salts" refer to pharmaceutically acceptable salts of a compound, which are derived from a variety of organic and inorganic counterions known in the art, such as sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and, if the molecule contains a basic functional group, include salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, and oxalate.

[0022] The terms “engine,” “engine antibiotic,” or “engine-containing cytotoxin” refer to any cytotoxin characterized by the presence of a 3-en-1,5-diine structural feature as part of a cyclic molecule known in the art, including neocardinostatin (NCS), C-1027, kedulcidin (KED), mazulopeptin (MDP), N1999A2, sporolide (SPO), cyanosporaside (CYA and CYN), as well as physiolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamemycin, shisimicin, and unicaramycin (UCM).

[0023] As used herein, the term "alkylamino sugar" means a tetrahydropyranyl moiety that is attached to an alcohol functional group via its 2-position, thereby forming an acetal functional group, and further substituted by at least one N-alkylamino group at its 3-, 4-, or 5-position. In this context, "N-alkylamino group" refers to an amino group having one methyl, ethyl, or 2-propyl group.

[0024] The term "click probe" refers to a functionalized moiety capable of producing a click reaction, i.e., two compatible click probes that click together so as to be covalently bonded 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, click probe Q in the compound according to the present invention can react with click probe F on a (modified) protein such that, upon occurrence of the click reaction, a conjugate is formed in which the protein is conjugated to the compound according to the present invention, where F and Q are compatible click probes.

[0025] In this specification, the "acylsulfamide moiety" is defined as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamolylated 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, and particularly in the examples, this group is also referred to as "HS".

[0026] A "coding sequence," or a sequence that "codes" an expression product, such as RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme; that is, the nucleotide sequence codes for the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence may include a start codon (usually ATG) and a stop codon.

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

[0028] The term “glycoprotein” is used herein in its usual scientific sense and refers to a protein containing one or more monosaccharides or oligosaccharides ("glycans") covalently linked to the protein. Glycans may be linked to hydroxyl groups on the protein (O-linked glycans), e.g., hydroxyl groups of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline; or to amide functional groups on the protein (N-glycoproteins), e.g., asparagine or arginine; or to carbon atoms on the protein (C-glycoproteins), e.g., tryptophan. A glycoprotein may contain two or more glycans; it may contain a combination of one or more monosaccharides and one or more oligosaccharide glycans; or it 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 are therefore eligible to be glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antarticalipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0029] The term "glycan" is used herein in its usual scientific sense and refers to a monosaccharide or oligosaccharide chain linked to a protein. Therefore, the term glycan refers to the carbohydrate portion of a glycoprotein. A sugar chain is linked to a protein via the C-1 carbon of a single sugar, either without further substitution (monosaccharide) or with further substitution of one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain 1 to about 10 sugar segments. However, if a longer sugar chain is linked to a protein, that chain is also considered a glycan herein. Glycans of glycoproteins can be monosaccharides. Typically, monosaccharide glycans of glycoproteins consist of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man), or fucose (Fuc) covalently linked to the protein. Glycans can also be oligosaccharides. Oligosaccharide chains of glycoproteins can be linear or branched. In oligosaccharides, the sugar directly linked to the protein is called the core sugar. In oligosaccharides, sugars that do not directly bind to a protein but bind to at least two other sugars are called internal sugars. In oligosaccharides, sugars that do not directly bind to a protein but do not have further sugar substituents on one or more of the other hydroxyl groups are called terminal sugars. To avoid misunderstanding, oligosaccharides in glycoproteins may have multiple terminal sugars, but there is only one core sugar. Glycans can be O-linked glycans, N-linked glycans, or C-linked glycans. In O-linked glycans, monosaccharides or oligosaccharide glycans bind to the oxygen atom in the amino acid of a protein, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, monosaccharides or oligosaccharide glycans bind to the protein via the nitrogen atom in the amino acid of a protein, typically via the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In C-linked glycans, monosaccharides or oligosaccharide glycans bind to the carbon atom in the amino acid of a protein, typically the carbon atom of tryptophan (Trp).

[0030] The term “antibody” (AB) is used herein in its ordinary scientific sense. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. An antibody is an example of a glycoprotein. The term “antibody” as used herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-stranded and single-stranded 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, e.g., cleaved 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] Antibodies can be natural or conventional antibodies, consisting of two heavy chains linked to each other by disulfide bonds, with each heavy chain linked to a light chain by disulfide bonds. There are two types of light chains: lambda(l) and kappa(k). The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains 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 heavy chain (VH) determine antigen binding recognition and specificity. The constant domains of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of immunoglobulin and consists of a variable region of one light chain and one heavy chain. Immunoglobulins can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgGA1, and IgGA2), 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 manipulated within the Fc domain to enhance or inactivate binding to the Fc-gamma receptor, as summarized by Saunders et al. Front.Immunol.2019,10,doi:10.3389 / fimmu.2019.01296 and Ward et al., Mol.Immunol.2015,67,131-141.For example, the combination of Leu234Ala and Leu235Ala (commonly called the LALA mutation) eliminates FcγRIIa binding. Elimination of binding to the Fcγ receptor can also be achieved by mutations in the N297 amino acid to any other amino acid except asparagine, mutations in the T299 amino acid to any other amino acid except threonine or serine, or, for example, by enzymatic deglycosylation or trimming of a fully glycosylated antibody by PNGase F or endoglycosidase. Immunoglobulins may be derived from any species, including 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 across a comparison frame, where the polynucleotide or polypeptide sequence portion of the comparison frame may contain additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. A sequence “at least 85% identical to a reference sequence” is a sequence that, in its entire length, has 85% or more sequence identity with the full length of the reference sequence, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0033] The term "CDR" refers to the complementarity-determining region, and antibody specificity lies in the structural complementarity between the antibody-binding site and the antigenic determinant. The antibody-binding site is primarily composed of residues derived from the hypervariable or complementarity-determining region (CDR). Occasionally, residues derived from the non-hypervariable or framework region (FR) affect the overall domain structure and, consequently, the binding site. Therefore, the complementarity-determining region, or CDR, refers to the amino acid sequence that together determines the binding affinity and specificity of the innate Fv region of the innate immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs: CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, a conventional antibody-antigen-binding site contains six CDRs, including sets of CDRs from the heavy chain V region and the light chain V region, respectively.

[0034] The terms “monoclonal antibody” or “mAb,” as used herein, refer to an antibody molecule consisting of a single amino acid sequence directed toward a specific antigen and should not be interpreted as requiring antibody production by any particular method. Monoclonal antibodies 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, the chimeric antibody contains the CH and CL domains of another antibody, and in one embodiment, the VH and VL domains of a non-human animal antibody associated with a human antibody. Any animal can be used as the non-human animal, such as a mouse, rat, hamster, or rabbit. A chimeric antibody may also exhibit multispecificity, having specificity for at least two different antigens.

[0036] The term "humanized antibody" refers to an antibody that is entirely or partially of non-human origin and has been modified to avoid or minimize the immune response 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, in most cases, the human CH and CL domains. A "fragment" of a (conventional) antibody contains a portion of an intact antibody, particularly 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. Conventional antibody fragments may also be heavy chain antibodies or single-domain antibodies such as VHH.

[0037] As used herein, "PTK7," also known as colon cancer kinase 4 (CCK4), represents a highly conserved member of the pseudokinase family of receptor tyrosine kinases, without observable kinase activity across various species. [Brief explanation of the drawing]

[0038] [Figure 1] This shows a typical set of reactive groups (F) that, when present in a biomolecule, yield a connecting group Z(1a-1h) upon reaction with reactive group Q. Functional groups F can be artificially introduced (manipulated) into biomolecules at any optimal position. [Figure 2] The following outlines how antibody conjugates can be obtained from any monoclonal antibody in a two-step process. In the first step, azide-modified UPD-Gal or UDP-GalNAc conjugates to a monoclonal antibody in a one-pot process involving (a) trimming of the glycan (to core GlcNAc) by endoglycosidase and (b) binding of the azide sugar under the action of glycosyltransferase (galactosyltransferase or its variant or GalNActransferase), thereby generating a β-glycoside 1-4 linkage between azide-modified GalNAc and GlcNAc. In the second step, the azide-modified antibody is reacted with appropriately functionalized cyclooctin to generate an antibody conjugate. [Figure 3] For example, the structures of several UDP sugar derivatives of galactosamine 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 an azide group (2d) at the 6-position are shown. [Figure 4] The following are cyclooctynes ​​(A-T) suitable for metal-free click chemistry, which are preferred options for the reactive part Q. [Figure 5]A set of exemplary toxic payloads for conjugation to various PTK7-targeted monoclonal antibodies according to the present invention is shown. Linker binding sites (to amino groups present in the payload) are indicated by arrows. A preferred conjugate according to the present invention contains a payload including the binding sites, as shown in Figure 5. [Figure 6] The structures of various camptothecins that are preferred payloads in the context of the present invention are shown. [Figure 7] The structures of BCN-linker drugs used to prepare ADCs via click chemistry to azide sugar remodeling antibodies, having the following payloads (3=exatecan, 4=MMAE, 5a=calicheamicin γ1 I, 5b=glycine-calicheamicin γ1 I, 6=6-aminohexanoyl-maytansinoid), are shown. [Figure 8A] This shows the binding of cofetuzumab to hPTK7 and cPTK7 after correcting for background noise. [Figure 8B] This demonstrates the binding of all cofetuzumab-based ADCs to hPTK7, equivalent to the corresponding mAb. [Figure 9] In vitro titer data for A431 cells treated with increased concentrations of cofetuzumab-4, cofetuzumab-5b, and the negative control B12-3 are shown. Both cofetuzumab ADCs resulted in cell death with IC50 values ​​of 3.9 nM and 0.1 nM, respectively. [Figure 10A] The in vivo efficacy data of PTK7 expressing the NCI-H446 tumor model over time are shown (Figure 10A: up to t=14, Figure 10C: up to t=44). 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) resulting in tumor growth delay. Other ADCs, including cofetuzumab-Aur0101, show only marginal tumor growth delay (similar to the isotype control ADC B12-3). [Figure 10B] The mouse body weight over time is shown (Figure 10B: up to t=14, Figure 10D: up to t=28 or 44). [Figure 10C] The in vivo efficacy data of PTK7 expressing the NCI-H446 tumor model over time are shown (Figure 10A: up to t=14, Figure 10C: up to t=44). 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) resulting in tumor growth delay. Other ADCs, including cofetuzumab-Aur0101, show only marginal tumor growth delay (similar to the isotype control ADC B12-3). [Figure 10D] The mouse body weight over time is shown (Figure 10B: up to t=14, Figure 10D: up to t=28 or 44). [Figure 11] The 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 monkey PTK7 relatively. 12C6a-3 shows no binding at all. 4D5-3 and 7C8-3 show similar binding to hPTK7 and cPTK7, but 7C8 clearly has a higher binding affinity to rPTK7. [Figure 12A] This report presents in vivo efficacy data for PTK7 expressing an NCI-H446 tumor model over time. 12C6-3 does not show tumor growth inhibition because it is vehicle-like. Both 7C8-3 and 4D5-3 show critical tumor growth delay. Cofetuzumab-3 shows tumor growth inhibition but also early tumor regrowth. 12C6-3 shows 99.7% tumor growth inhibition by day 40. [Figure 12B] This shows the mouse's body weight over time. [Figure 12C] This shows the survival over time in a Kaplan-Meier plot of the NCI-H446 model. [Figure 13A]This report presents in vivo efficacy data of PTK7 expressing a BR1282 PDX tumor model over time. 12C6-3 exhibits a better tumor growth inhibition rate than all 7C8-3, 4D5-3, and 12C6a-3, and even compared to cofetuzumab-Aur0101. Compared to cofetuzumab-3, both ADCs show a similar response and exhibit similarly good tumor growth inhibition. [Figure 13B] This shows the mouse's body weight over time. [Figure 14A] This report presents in vivo efficacy data for PTK7 expressing an NCI-H446 tumor model over time. Olaparib as a single treatment did not show a significant effect. Both 12C6-3 and 12C6-9 showed tumor growth inhibition / stagnation. However, the combination of olaparib and 12C6-3 showed a clear additive effect, resulting in a complete response in 6 out of 8 mice at day 21, and the study is still ongoing. [Figure 14B] This shows the mouse's body weight over time. [Figure 15] The in vivo tolerability data for 12C6-3 in female Sprague Dawley rats over time are shown. No toxicity or weight loss was observed at any concentration. Therefore, the maximum tolerable dose exceeds 150 mg / kg. [Modes for carrying out the invention]

[0039] In a first embodiment, 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 that can target PTK7-expressing tumors. -L is a linker that connects Z to D, -Z is a connecting group, -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L7 ) w’ - wherein G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w’ is 0, 1, or 2, 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, hemiasterlins, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBD), and analogs or prodrugs thereof, - b is 0 or 1, - x is 1 or 2, - y is 1, 2, 3, or 4.

[0040] Salts of the antibody conjugate according to structure (1), preferably pharmaceutically acceptable salts, 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, which comprises 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 that can react with Q in a conjugation reaction, and Q and F react to form a linking group Z. In this reaction, the conjugate described in general structure (1) is formed. The process according to this aspect relates to the following bioconjugation reaction: AB-[(L 6 ) b -{F} x ) y + Z-L-D → AB-[(L 6 ) b -{Z-L-D} x ) y (3) (2) (1)

[0042] Below, we first define the antibody conjugate following structure (1). The structural characteristics of the antibody conjugate following structure (1) also apply to the compound following structure (2) and the antibody following structure (3), because they remain unchanged in the conjugation reaction, except for the reactive parts F and Q which are converted to the linking group Z during the reaction between the compound following structure (2) and the antibody following structure (3).

[0043] In a third embodiment, the present invention relates to an applicable antibody conjugate according to structure (1) for targeting PTK7-expressing cells. In connection therewith, the present invention relates to a first medical use and a second medical use of the antibody conjugate according to structure (1).

[0044] As will be understood by those skilled in the art, the definitions of chemical parts and their preferred embodiments apply to all aspects of the present invention.

[0045] Antibody conjugate of general structure (1) In a first embodiment, 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 that can target PTK7-expressing tumors. -b is either 0 or 1. -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - is a monosaccharide, where G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, L 7 These are -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. -Z is a connecting group, -L is a linker that connects Z to D, -D is selected from the group consisting of anthracyclines, camptothecin, tubulisin, engine, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, BCL-XL inhibitors, hemiasterin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs. -x is either 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 treatment. The term "expressing" is commonly used in the art and refers to the overexpression of a target in healthy tissue. An antibody capable of targeting PTK7-expressing tumors is also called an anti-PTK7 antibody, a PTK7-targeted antibody, or a PTK7-conjugated antibody. The 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 region of these antibodies may have one or more mutations, such as 0 to 10 mutations or 0 to 5 mutations. Mutations that alter binding to the FcRn receptor are particularly preferred to regulate the antibody's half-life. For example, by including mutations from Met to Tyr, Ser to Thr, and Thr to Glu in the region of amino acids 254-260 of the heavy chain, often called YTE, in IgG1 Fc, the antibody's binding to human FcRn is increased by approximately 11 times, thereby increasing the circulating half-life by approximately 3.5 times. For example, the YTE mutations in cofetuzumab are Met254Tyr, Ser256Thr, and Thr258Glu. Thus, in one embodiment, the antibody has a half-life of 2.5 × 10⁻⁶. -6 Less than M, preferably 0.05 to 0.99 × 10-6 Within the range of M, more preferably 0.1 to 0.49 × 10 -6 Within the range of M, most preferably 0.2 to 0.4 × 10 -6 Apparent human FcRn binding affinity K within the M range D,app It has. Apparent binding affinity K D,app This can be determined according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, antibody AB is a YTE mutation of the preferred antibody as defined above or below.

[0048] In a preferred embodiment, the antibody is cofetuzumab. Cofetuzumab may also be defined to include a light chain sequence according to SEQ ID NO: 8 and a heavy chain sequence according to SEQ ID NO: 7, 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 exatecan as payload D.

[0049] In a preferred embodiment, the antibody is 12C6. 12C6 may also be defined to include 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 a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably 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 sequence identity being 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 exatecan as payload D.

[0051] In a preferred embodiment, the antibody is 4D5. 4D5 may also be defined to include 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 calicheamycin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamycin, MMAE, and exatecan, and most preferably exatecan as payload D.

[0052] In a preferred embodiment, the antibody is 7C8. 7C8 may also be defined to include 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 calicheamycin, MMAE, PF-06380101, exatecan, and DXd, more preferably a payload D selected from the group consisting of calicheamycin, MMAE, and exatecan, and most preferably exatecan as payload D.

[0053] In a preferred embodiment, the antibody is Hu23. Hu23 may also be defined to include 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 exatecan as payload D.

[0054] In a preferred embodiment, the antibody is Hu58. Hu58 may also be defined to include 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 a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, and most preferably exatecan as payload D.

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

[0056] The preferred antibodies are those V L Domain and V HDefined by domains, these together form a variable domain that binds to the antigen. Therefore, in a preferred embodiment, the antibody is selected from the group consisting of SEQ ID NOs: 2, 6, 9, 11, 14, 18, 21, 24, 32, and 36. L A domain and V selected from the group consisting of sequence numbers 1, 5, 10, 12, 13, 17, 23, 30, and 34. H The domain is included, and the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85%, at least 90%, or at least 95%, and most preferably at least 99% or 100%. In a particularly preferred embodiment, the antibody is V of SEQ ID NO: 6 L Domain and V of sequence number 5 H The domain is included, and the sequence identity is at least 70%, preferably at least 75%, or at least 80%, more preferably at least 85%, or at least 90%, or at least 95%, most preferably at least 99%, or even more preferably 100%.

[0057] The aforementioned sequence identity is V L Domain and V H This refers to the complete sequence of the domain. While the entire sequence of these domains allows for some mutations within the sequence without compromising binding to PTK7, the complementarity-determining region (CDR) preferably has higher sequence identity to ensure that binding to PTK7 is not significantly compromised. The location of the CDR is shown in the table below. Therefore, the antibody is selected from the group consisting of SEQ ID NOs: 2, 6, 9, 11, 14, 18, 21, 24, 32, and 36. L A domain and V selected from the group consisting of sequence numbers 1, 5, 10, 12, 13, 17, 23, 30, and 34. H The domain and preferably V of sequence number 6. L Domain and V of sequence number 5 H Preferably, the domain is included, and the sequence identity of the CDR is at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably 100%. Those skilled in the art will understand the V specified above.L Domain and V H It is understood that the domain can be combined with a suitable constant domain to form a complete antibody.

number

number

[0058] In a particularly preferred embodiment, the antibody is V of SEQ ID NO: 2 L Domain and V of sequence number 1 H Domain and, or V of Sequence ID 6 L Domain and V of sequence number 5 H Domain and, or V of sequence number 9 L Domain and V of sequence number 10 H Domain and, or V of sequence number 11 L Domain and V of sequence number 12 H Domain and, or V of sequence number 14 L Domain and V of sequence number 13 H The domain and, or V of sequence number 18 or 21 L Domain and V of sequence number 17 H Domain and, or V of sequence number 24 L Domain and V of sequence number 23 H Domain and, or V of sequence number 32 L Domain and V of sequence number 30 H Domain and, or V of sequence number 36 L Domain and V of sequence number 34 H This includes the domain. In this specification, the sequence identity defined above applies to the complete sequence and to the CDR.

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

[0060] The aforementioned sequence identity refers to the complete sequences of the light and heavy chains. While the entire sequences of these chains allow for some mutations within the sequence without compromising binding to PTK7, the CDR preferably has higher sequence identity to ensure that binding to PTK7 is not significantly compromised. The location of the CDR is shown in the table below. Therefore, the antibody preferably comprises a light chain selected from the group consisting of SEQ ID NOs: 4, 8, 16, 20, 22, 26, 33, and 37, and a heavy chain selected from the group consisting of SEQ ID NOs: 3, 7, 15, 19, 25, 31, and 35, with the CDR having sequence identity of 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 the light chain of SEQ ID NO: 4 and the heavy chain of SEQ ID NO: 3, or the light chain of SEQ ID NO: 8 and the heavy chain of SEQ ID NO: 7, or the light chain of SEQ ID NO: 16 and the heavy chain of SEQ ID NO: 15, or the light chain of SEQ ID NO: 20 or 22 and the heavy chain of SEQ ID NO: 19, or the light chain of SEQ ID NO: 26 and the heavy chain of SEQ ID NO: 25, or the light chain of SEQ ID NO: 33 and the heavy chain of SEQ ID NO: 31, or the light chain of SEQ ID NO: 37 and the heavy chain of SEQ ID NO: 35. In this specification, the sequence identity defined above applies to the complete sequence and to the CDR.

[0062] Linker L 6 If the reactive group F is directly attached to the antibody, or even to a part of the antibody structure, a linker L connects 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 It does not exist, and b=0. This is the case, for example, in cysteine ​​conjugation and lysine conjugation. Alternatively, linker L connects AB to F (for antibody of structure (3)) or AB to Z (for conjugate of structure (1)). 6 The reactive group F can also be introduced into the antibody using L 6 L exists, and b=1. 6 If present, the reactive group F is typically introduced into the glycan of this antibody. This is the case of conjugation via an artificially introduced reactive group F, for example, by using transglutaminase or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase). For example, modified sugar residue S(F) x The glycan may be extended by a single monosaccharide residue S that is introduced into the glycan to introduce x reactive groups F onto the antibody glycan. In the most preferred embodiment, the conjugation occurs via the antibody's sugar chain and b=1. The site of the conjugation is preferably in the antibody's heavy chain.

[0063] If present, L 6This is a linker that connects AB to F or Z, and -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ Represented by -, where G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, L 7 These are -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. Typically, L 6 The linker L is formed at least partially by the antibody glycan. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at or near position 297 of the heavy chain, which is modified by a complex-type glycan. While this naturally occurring glycosylation site of the antibody is preferably used, other glycosylation sites, including those introduced artificially, can also be used for linker L. 6 It can be used for the connection of L. Therefore, in a preferred embodiment, 6 It is attached to an amino acid of the antibody located within the range of 250 to 350 on the heavy chain, preferably within the range of 280 to 310 on the heavy chain, more preferably within the range of 295 to 300 on the heavy chain, and most preferably at position 297 on the heavy chain.

[0064] L 6 -GlcNAc(Fuc) w -(G) j - is the antibody glycan, or a part thereof. Therefore, the glycan -GlcNAc(Fuc) w -(G) j- is typically derived from the original antibody, GlcNAc is the N-acetylglucosamine moiety, and Fuc is the fucose moiety. Fuc is typically linked to GlcNAc via an α-1,6-glycosidic bond. Usually, the antibody may be fucosylated (w = 1) or not (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 can also be referred to as the core-GlcNAc residue, is a monosaccharide that binds directly to the peptide portion of the antibody.

[0065] S may be directly connected to the core-GlcNAc(Fuc) w moiety, i.e., j may be 0, which means that before S binds, the rest of the glycan is removed from the core-GlcNAc(Fuc) w moiety. Such trimming of the glycan is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, one or more monosaccharide residues may be present between the core-GlcNAc(Fuc) w moiety and S, i.e., j is an integer within 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 within the range of 2 to 5. (G) j is typically linked to GlcNAc(Fuc) via a β-1,4 bond wIt is connected to the GlcNAc portion. In a preferred embodiment, j is 3, 4, or 5. Any monosaccharide that may be present in the 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 show little to no binding to the Fc-gamma receptor, while antibody conjugates with j in the range of 4 to 10 do bind to the Fc-gamma receptor. Therefore, by selecting a specific value for j, a desired degree of binding to the Fc-gamma receptor can be obtained. Therefore, the antibody is trimmed to j=0, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably j=0, 3, 4, or 5, most preferably j=0.

[0066] S is a sugar or a sugar derivative. The term "sugar derivative" is used herein to refer to a monosaccharide sugar, i.e., a derivative of a monosaccharide sugar containing substituents and / or functional groups. Preferred 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 known as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Preferably, S is selected from Glc, Gal, GlcNAc, and GalNAc. In a particularly preferred embodiment, S is GalNAc.

[0067] x is an integer representing the number of linking groups Z (of conjugate (1)) or reactive groups F (of antibody (3)) that are attached to sugar (derivative) S. Thus, the antibody according to the invention contains a moiety S that includes x reactive moieties F. Each of these reactive moieties F reacts with the 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 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 a linker L 7 may be present between S and Z or F. L 7 is a linker that links S to Z. L 7 may be present (w’ = 1 or 2), or may not be present (w’ = 0). Typically, each moiety Z may be connected to S via a linker L 7 and thus, in one embodiment, w’ = 0 for x. Preferably, L 7 is not present and each binding site Z is directly attached to S. When present, L 7 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 having x reactive groups F or being connected to x connecting groups Z, which are attached to the antibody. y is 1, 2, 3, or 4, preferably y = 2 or 4, most preferably y = 2. Thus, the antibody contains y moieties S each of which includes x reactive moieties F. Each of these reactive moieties F reacts with the reactive moiety Q of a compound according to general structure (2) such that x + y linking groups Z are formed and x + y compounds according to general structure (2) are attached to a single antibody. Each compound according to general structure (2) may, for example, have a nitrogen atom N in L* Due to branching, it may contain multiple payloads. Each compound according to the general structure (2) preferably contains one or two appearances of D, most preferably two appearances of D. In a particularly preferred embodiment, linker L 1 The second appearance of D is connected to the branched nitrogen atom N. * It contains.

[0070] The amount of payload (D) molecules bound to a single antibody is known in the art as the DAR (drug-antibody ratio). In the context of this invention, the DAR is preferably an integer in the range of 1 to 8, more preferably 2 or 4, and most preferably DAR=4. Alternatively, the DAR is preferably an integer in the range of (x+y)~[(x+y)×2], and most preferably DAR=[(x+y)×2]. For preferred values ​​of 1 for x and 2 for y, the DAR is preferably 4. These are theoretical DAR values, and it will be understood that in practice, the DAR may deviate slightly from these values ​​due to imperfect conjugation. Typically, the conjugate is obtained as a stochastic mixture of antibody-drug conjugates, and the DAR values ​​vary among the 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). In the case of such a mixture, the DAR often refers to the average DAR of the mixture. This is well known in the field of bioconjugation. However, conjugation occurs via glycans (i.e., L at b=1). 6 If (i.e., if

[0071] Conjunction Z Z is a connecting group that covalently links both parts of the conjugate according to the present invention. The term “connecting group” as used herein refers to a structural element resulting from a reaction between Q and F that connects one part of a conjugate to 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. For example, when the carboxyl group of RC(O)-OH reacts with the amino group of H2N-R' to form RC(O)-N(H)-R', R is connected to R' via the connecting group Z, and Z can be represented by the group -C(O)-N(H)-. Since the connecting group Z originates from a reaction between Q and F, it can take any form.

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

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

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

[0075] For example, if F contains an amino group or is an amino group, the complementary group Q contains a ketone group and an activated ester group, and the corresponding connecting group Z is as shown in Figure 1.

[0076] For example, if F contains a ketone group or is a ketone group, the complementary group Q contains an (O-alkyl)hydroxylamino group and a hydrazine group, and the corresponding connecting group Z is as shown in Figure 1.

[0077] For example, if F contains an alkynyl group or is an alkynyl group, the complementary group Q contains an azide group, and the corresponding connecting group Z is as shown in Figure 1.

[0078] For example, if F contains an azide group or is an azide group, the complementary group Q contains an alkynyl group, and the corresponding connecting group Z is as shown in Figure 1.

[0079] For example, if F contains or is a cyclopropenyl group, a transcyclooctene group, or a cyclooctin group, the complementary group Q contains a tetradinyl 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 producing dihydropyridazine (from reaction with an alkene) or pyridazine (from reaction with an alkyne).

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

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

[0082] In a first preferred embodiment, Z is formed by cycloaddition. Preferred cycloadditions are (4+2)-cycloaddition (e.g., Diels-Alder reaction) or (3+2)-cycloaddition (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron-required Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition, more preferably an alkyne-azide cycloaddition, is used, most preferably Q is an alkyne group or contains an alkyne group and F is an azide group. Cycloadditions such as the Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art and those skilled in the art know 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 group. The triazole moiety is particularly preferred to be present in Z. In one embodiment, Z is formed from Q comprising a (hetero)cycloalkene moiety, i.e., a (hetero)cycloalkyne moiety. In an alternative embodiment, Z is formed from Q comprising a (hetero)cycloalkane moiety, i.e., a (hetero)cycloalkene moiety. In a preferred embodiment, Z has structure (Z1). [ka]

[0084] Here, the bond indicated by --- is either a single bond or a double bond. Furthermore, -Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za) to (Zj) as defined below. ** The carbon atoms labeled with correspond to the two carbon atoms in the bond shown as --- of ring Z condensed (Z1), -R15 These are, independently, hydrogen, halogen, -OR 16 NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These are linked together to form an optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero) substituent, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -Y 2 C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31 And in the formula, S (+) B (-) A cationic sulfur atom that is canceled out by, in the formula, B (-) is an anion, and each R 31 R 15 Alternatively, it is 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 8 to 16. -Ring A is formed by cycloaddition and is preferably selected from (Za)-(Zj).

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

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

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

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

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

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

[0091] Therefore, in a preferred embodiment, Z includes a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptine moiety according to structure (Z37) is not substituted.

[0092] In a preferred embodiment, Z includes a (hetero)cyclooctene moiety following structure (Z8), more preferably a (hetero)cyclooctene moiety following structure (Z29), which is optionally substituted. Preferably, the cyclooctene moiety following structure (Z8) or (Z29) is not substituted. In the context of this embodiment, Z preferably includes a (hetero)cyclooctene moiety following structure (Z39) shown below, where V is (CH2) lThereafter, 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 base (Z39), l is most preferably 1. Most preferably, Z follows a structure (Z42) further defined below.

[0093] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety conforming to the structures (Z26), (Z27), or (Z28), which are optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety conforming to the structures (Z40) or (Z41) shown below, where Y 1 is O or NR 11 And in the formula, R 11 These are, independently, hydrogen, linear or branched C1-C 12 Alkyl alkyl groups, or C4-C 12 Selected from the group consisting of (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 follows structure (Z43), which is further defined below.

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

[0095] In a particularly preferred embodiment, Z comprises a cyclooctenyl group and follows the structure (Z42). [ka] Here, -*The bond labeled with is connected to S, ** The wavy coupling labeled with is connected to L, -R 15 These are, independently, hydrogen, halogen, -OR 16 NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These are linked together to form an optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero) substituent, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -R 18 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -R 19 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group is optionally interrupted by one of several 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 This is a second appearance of Z (or Q) or D connected via the spacer portion, -l is an integer in the range of 0 to 10.

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

[0097] In a particularly preferred embodiment, Z comprises a (hetero)cyclooctinyl group and follows the structure (Z43). [ka] (Z43) Here, -* The bond labeled with is connected to S, ** The wavy coupling labeled with is connected to L, -R15 These are, independently, hydrogen, halogen, -OR 16 NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These are linked together to form an optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero) substituent, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -Y is N or CR 15 That is the case.

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

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

[0100] In an alternative preferred embodiment, Z includes a (hetero)cycloalkane moiety, i.e., the bond indicated as --- is a single bond. The (hetero)cycloalkane group may also be called a heterocycloalkanyl group or a cycloalkanyl group, preferably a cycloalkanyl group, and 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 a (hetero)cyclopropanyl group, a (hetero)cycloheptanyl group, or a (hetero)cyclooctanyl group, and the (hetero)cyclopropanyl group, a trans-(hetero)cycloheptanyl group, or a (hetero)cyclooctanyl group is optionally substituted. Preferably, Z includes 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). Here, Y 3 C(R 23 )2, NR 23 , or O is selected, and each R 23The bonds are individually hydrogen, C1-C6 alkyl, or optionally connected to L via spacers, and the bonds labeled --- are single or double bonds. In a further preferred embodiment, the cyclopropanyl group follows structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group follows structure (Z50) or (Z51). In yet another preferred embodiment, the (hetero)cyclooctane group follows structure (Z52), (Z53), (Z54), (Z55) or (Z56). [ka]

[0101] Here, the R group(s) on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. The 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 ring (Z44)~(Z56) into which ring Z is condensed. * The carbon labeled with is directly attached to the antibody peptide chain. The preferred ring Z is selected from (Zo) to (Zr). Since the connecting group Z is formed by a reaction with a (hetero)cycloalkene in the context of this embodiment, the bond shown above as --- is a single bond. [ka]

[0102] In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably nucleophilic substitution or Michael addition, preferably Michael addition. A preferred Michael reaction is a thiol-maleimide dilation, most preferably where 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 succinic acid amide derivative thereof. Preferred choices for the connecting group Z include portions selected from (Z57) to (Z71) shown below. [ka]

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

[0104] In a preferred embodiment, the connecting group Z includes a portion selected from (Z1) to (Z71).

[0105] Linker L Linker L connects payload D to linking group Z (in a conjugate according to structure (1)) or to reactive group Q (in a compound according to structure (2)). The linker is known in the art and may be cleavable or incleavable. Linker L preferably includes a self-sacrificing 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 It is connected to payload D, (L 1 ) n Structure - (L 1 ) n- (L 2 ) o- (L 3 ) p- (L 4 ) q - as linker L. Here, L 1 , L 2 , L 3 , and L 4 is a linker or connecting unit, where 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 linker L 1 and L 2 There exists (i.e., n=1, o=1, p=0 or 1, q=0 or 1), more preferably linker L 1 , L 2 , and L 3 L 4 The linker L is either 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 There exists (i.e., n=1, o=1, p=1, q=1). In one embodiment, linker L 1 , L 2 , and L 3 L 4It does not exist (i.e., n=1, o=1, p=1, q=0).

[0107] Linker, especially Linker L 1 The conjugate may include one or more branching points for joining multiple payloads to a single conjugate. In a preferred embodiment, the linker of the conjugate according to the present invention includes a branching portion. In the context of the present invention, “branching portion” refers to a portion embedded in the linker that connects the three parts. In other words, the branching portion includes at least three connections to the other parts, typically one connection to Z or Q, one connection to payload D, and one connection to a second payload D. The branching portion, if present, is preferably on linker L 1 A more preferable Sp 2 In part, or NR 13 It is embedded as a nitrogen atom. Any portion containing at least three bonds to other portions is suitable as a branched portion in the context of the present invention. In preferred embodiments, the branched portion is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)ring, or a polycyclic portion. Most preferably, the branched portion is a nitrogen atom.

[0108] Linker L 1 Linker L 1 It either does not exist (n=0) or it exists (n=1). Preferably, linker L 1 L exists, and n=1. 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 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group, C9~C 200The group can be selected from the group consisting of arylalkylene groups.Optionally, alkylene groups, alkenylene groups, alkylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups, and arylalkylene groups may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, wherein the heteroatoms are preferably O, S(O) y’ , and NR 21 Selected from the group consisting of, where y' is 0, 1, or 2, preferably y'=2, R 21 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups.

[0109] In a preferred embodiment, linker L 1 It contains polar groups. 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 (wherein z' is the number of carbon atoms in the alkane, preferably z'=1 to 10), -(O) a -C(O)-NH-S(O)2-NR 13 -(See structure (23) as further defined below), -C(S(O)3 (-) )-,-C(C(O)2 (-) )-, -S(O)2-, -P(O)2 (-) -, -O(CH2CH2O) t -, -NR 30 (CH2CH2NR 30 ) t - and the following two structures can be selected. [ka]

[0110] The polar group may also preferably include an amino acid selected from Arg, Glu, Asp, Ser, and Thr. Here, a and R 13 The structure (23) is further defined below. t is an integer within the range of integers 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 These are H, C individually. 1~12 Alkyl, C1~ 12 Ariel, C 1~12 Alkaline, or C 1~12 It is Aralkir. Linker L 1 It may contain two or more such polar groups, such as at least two polar groups. The polar groups also branch the linker L that branches the branched portion defined elsewhere. 1 It may be present in the branching. Preferably, nitrogen or carbon atoms are used as the branching portion. -O(CH2CH2O) is present in the branching portion. t - The presence of polar groups is particularly preferable.

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

[0112] The dashed line represents the remainder of the compound, typically Q and L. 2 , L 3 , L 4 Or D, preferably Q and L 2 Represents a connection to (O). Preferably, (O) a The C(O) part is connected to Q, NR 13 The part is L 2 , L 3 , L 4 Or D, preferably L2 It connects to the network.

[0113] In structure (23), a=0 or 1, preferably a=1, and R 13 is hydrogen, C 1~ C 24 Alkyl alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (hetero)aryl group, C 3~ C 24 Alkyl (hetero)aryl group, and C 3~ C 24 Selected from the group consisting of (hetero)arylalkyl groups, C 1~ C 24 Alkyl alkyl group, C 3~ C 24 Cycloalkyl groups, C 2~ C 24 (hetero)aryl group, C 3~ C 24 Alkyl (hetero)aryl group, and C 3~ C 24 (Hetero)arylalkyl groups are optionally substituted with O, S, and NR. 14 Optionally interrupted by one or more heteroatoms selected from R 14 These are, independently, hydrogen and C 1~ Selected from the group consisting of C4 alkyl groups, or R 13 The spacer portion is preferably defined as Sp 2 It is connected to N via and in one embodiment, D is -(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 It is an alkyl group, more preferably R 13 is hydrogen or C1~C 16 It is an alkyl group, and more preferably, R 13 is hydrogen or C1~C 10It is an alkyl group, and the alkyl group is optionally substituted with O, S, and NR. 14 One or more heteroatoms selected from, preferably O, optionally interrupted by R 14 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In a preferred embodiment, R 13 is hydrogen. In another preferred embodiment, R 13 C1~C 20 Alkyl groups, fuaC1~C 16 Alkyl groups, more preferably C1-C 10 The alkyl group is optionally interrupted by one or more oxygen atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is more preferably a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 R 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, and even more preferably from the group consisting of hydrogen, methyl, and ethyl. 13 is hydrogen or methyl, most preferably R 13 It is hydrogen.

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

[0116] Here, a and R 13 As defined above, Sp 1 and Sp 2 The spacer portion is independent, 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, spacer Sp 1and Sp 2 These 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 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group, and C9~C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, arylalkylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylene group are optionally substituted with O, S, and NR 16 The expression is optionally interrupted by one or more heteroatoms selected from the group, where R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group, and C3~C 24 The group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted. When an alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylynylene group is interrupted by one or more heteroatoms as defined above, it is preferable that the group is interrupted by one or more oxygen atoms and / or one or more SS groups.

[0117] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently 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 Cycloalkylene group, C7~C 100 Alkyl arylene group, C7~C 100 Arylalkylene group, C8~C 100 Arylalkenylene group, and C9~C 100 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, arylalkylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylene group are optionally substituted with O, S, and NR 16 The expression is optionally interrupted by one or more heteroatoms selected from the group, where R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group, and C3~C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0118] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently 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 Cycloalkylene group, C7~C 50 Alkyl arylene group, C7~C 50 Arylalkylene group, C8~C 50 Arylalkenylene group, and C9~C 50Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, arylalkylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylene group are optionally substituted with O, S, and NR 16 The expression is optionally interrupted by one or more heteroatoms selected from the group, where R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group, and C3~C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0119] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently 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 Cycloalkylene group, C7~C 20 Alkyl arylene group, C7~C 20 Arylalkylene group, C8~C 20 Arylalkenylene group, and C9~C 20 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, arylalkylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylene group are optionally substituted with O, S, and NR 16 The expression is optionally interrupted by one or more heteroatoms selected from the group, where R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24Alkenyl group, C2~C 24 Alkynyl group, and C3~C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

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

[0121] Most preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently linear or branched C1-C 20 Selected from the group consisting of alkylene groups, the alkylene groups are optionally substituted with O, S, and NR. 16 The expression is optionally interrupted by one or more heteroatoms selected from the group, where R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group, and C3~C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted. In this embodiment, the alkylene group is unsubstituted, and O, S, and NR 16 , preferably optionally interrupted by one or more heteroatoms selected from the group O and / or SS, R 16 It is more preferable that these are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0122] Therefore, preferred spacer portion 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 The formula includes -, where r is an integer in the range of 1 to 50, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, and even more preferably 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, preferred linker L 1 is, -(W) k -(A) d -(B) e -(A) f -(C(O)) g - can be expressed as, in the formula, -d=0 or 1, preferably d=1. -e = 0 to 10, preferably an integer in the range of e = 0, 1, 2, 3, 4, 5, or 6, preferably an integer in the range of 1 to 10, most preferably e = 1, 2, 3, or 4. -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 either a -CH2-CH2-O- or -O-CH2-CH2- part, or (B) e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 -CH2- is the part where e1 is defined in the same way 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 It is C(O)NH-, and preferably W is -OC(O)NH-, -C(O)(CH2) m The molecule is 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, most preferably m=2 or 3. - Preferably, L 1 (W) k via Q, and (C(O)) g L via, preferably via C(O) 2 , L 3 , L 4 , or D, preferably L 2 It connects to the network.

[0124] In the context of this embodiment, the wavy line in structure (23) is (W) k (B) e , and (C(O)) g This represents connections to adjacent groups such as A. Preferably, A follows structure (23), where a=1 and R 13 =H or C1~C 20 Alkyl group, fuaR 13 =H or methyl, most preferably R 13 = H.

[0125] Preferred linker L 1 is structure-(W) k -(A) d -(B) e -(A) f -(C(O)) g - has, in the formula, (a) k=0, d=1, g=1, f=0, B=-CH2-CH2-O-, 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-, 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, linker L 1 It contains branched nitrogen atoms, which are Q or Z and (L 2 ) o It is located in the skeleton between and includes a further fraction D as a substituent, where D is preferably linked to a branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR in structure (23). 13 And in the formula, R 13 This connects to the second appearance of D via the spacer portion. Alternatively, the branched nitrogen atom is structure-(W) k -(A) d -(B) e -(A) f -(C(O)) g -according to L 1 It may be located inside. 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 is expressed as, where A, B, W, d, e, f, g, and k are as defined above, and each occurrence is individually selected, N * is, -(A) d -(B) e -(A) f -(C(O)) g’ -Two examples are linked branched nitrogen atoms. Here, both (C(O)) g’ The part is -(L 2 ) o -(L 3 ) p -(L 4 ) q -Connected to D, in the formula, L 2 , L 3 , L 4o, p, q, and D are as defined above and are selected individually. In a preferred embodiment, L 2 , L 3 , L 4 Each of , o, p, q, and D is (C(O)) g The same applies to both parts that are connected to it.

[0127] Preferred linker L containing branched nitrogen atoms 1 is structure-(W) k -(A) d -(B) e -(A) f -(C(O)) g -N * [-(A') d’ -(B') e’ -(A') f’ -(C(O)) g” -]2 is present, in the formula, (i) k=d=g=e'=1, f=d'=g'=0, W=-C(O)-, B=B'=-CH2-CH2-O-, and A follows 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-, and A follows 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 It either does not exist (o=0) or it exists (o=1). Preferably, linker L 2 It exists, and o=1. Linker L 2 This is a peptide spacer. The peptide spacer is preferably (NH-CR 17 -CO) n Defined by, in the formula, R 17represents an amino acid side chain known in the art. In this specification, amino acids may be natural or synthetic amino acids. Preferably, all amino acids are in their L configuration. n is an integer in the range of 1 to 5, preferably in the range of 2 to 5. Therefore, the peptide spacer preferably contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), a tripeptide (n=3), or a tetrapeptide (n=4), and most preferably, the peptide spacer is a dipeptide. Any peptide spacer may be used, but 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 , selected from 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 selected from Val-Cit or Val-Ala. In this specification, AcLys is acetyllysine and iGlu is isoglutamate. In one embodiment, L 2 =Val-Cit. In one embodiment, L 2 =Val-Ala

[0129] R 17is preferably an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolicine, 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 Preferably, it is selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp), and CH2(1H-indole-3-yl)(Trp). 17 Particularly preferred embodiments include CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). Most preferably, R 17 These are 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 17This is as defined above, and preferably R 17 This is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line is (L 1 ) n and (L 3 ) p This indicates a connection to, preferably L according to structure (L3). 2 (L 1 ) n to, and via C(O) (L 3 ) p It connects to the network.

[0132] Linker L 3 Linker L 3 It either does not exist (p=0) or it exists (p=1). Preferably, linker L 3 It exists and p=1. Linker L 3 This is a self-cutting spacer, also known as a self-sacrificing spacer. Preferably, L 3 This is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative following structure (L4). [ka]

[0133] Here, the dashed line represents Q or Z, L 1 or L 2 , and L 4 Or it indicates connection to D. Typically, PABC derivatives connect to Q, Z, and L via NH. 1 , or L 2 Preferably L 2 L via O 4 Alternatively, it connects to D.

[0134] A is a five-membered or six-membered aromatic or heteroaromatic ring, preferably a six-membered aromatic or heteroaromatic ring. Preferred five-membered rings are oxazole, thiazole, and furan. Preferred six-membered rings are phenyl and pyridyl. In preferred embodiments, 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 group, C3~C 10 Alkyl (hetero)aryl groups, and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 28 Optionally interrupted by one or more heteroatoms selected from R 28 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, 26 C3~C 10 It is a (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H is the most preferred polyalkylene glycol, preferably polyethylene glycol, preferably -(CH2CH2O) s H is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s = 1, 2, 3, or 4. More preferably R 21 is H or C(O)R 26 And in the formula, R 26 =4-methylpiperazine or morpholine. Most preferably, R 21 H is H.

[0136] Linker L 4 Linker L 4 It either does not exist (q=0) or it exists (q=1). Preferably, linker L 4 The linker L exists, and q=1. 4 teeth, -Structure-NR 22 -(C z -Alkylene)-C(O)- aminoalkanoic acid spacer (wherein z is an integer in the range of 1 to 20, R 22 (is H or C1-C4 alkyl), -Structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 -C(O)- Ethylene glycol spacer according to the formula (wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, R 22 (is H or C1-C4 alkyl), and -Structure-NR 22 -(C z -Alkilen)-NR 22 -(C(O)) h - Diamine spacer according to the formula (wherein h is 0 or 1, z is an integer in the range of 1 to 20, R 22 (is selected from H or C1-C4 alkyl).

[0137] Linker L 4 This is an aminoalkanoic acid spacer, i.e., -NR 22 -(C z The formula may be -alkylene)-C(O)-, where z is an integer in the range of 1 to 20, preferably 1 to 10, most preferably 1 to 6. In this specification, the aminoalkanoic acid spacer is typically L via a nitrogen atom. 3 It is connected to D via the carbonyl portion. Preferred linker L 4 L is selected from 6-aminohexanoic acid (Ahx, z=5), β-alanine (z=2), and glycine (Gly, z=1), and more preferably 6-aminohexanoic acid or glycine. In one embodiment, L4= It is 6-aminohexanoic acid. In one embodiment, L 4 = Glycine. In this specification, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 H is H.

[0138] Alternatively, Linker L 4 is structure-NR 22- (CH2-CH2-O) e6- (CH2) e7- (It may also be an ethylene glycol spacer according to the formula C(O)-, where e6 is an integer in the range of 1 to 10, preferably in the range of 2 to 6, and e7 is an integer in the range of 1 to 3, preferably e7 is 2. In this specification, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 H is H.

[0139] Alternatively, Linker L 4 is structure-NR 22 -(C z -Alkilen)-NR 22 -(C(O)) h -The diamine spacer may also conform to the formula, 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, more preferably z=2 or 5, and most preferably z=2. 22 is H or C1-C4 alkyl. In this specification, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is methyl. In this specification, h is preferably 1, in which case linker L 4 This is particularly suitable for conjugation via phenol hydroxyl groups present on payload D.

[0140] Payload D D represents a target molecule D that is attached to or to an antibody, also referred to in the art as a payload. D is selected from the group consisting of anthracyclines, camptothecin, tubulisin, engine, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, BCL-XL inhibitors, hemiasterin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs. Alternatively, D is defined as a pharmaceutically active substance such as an anticancer agent, preferably a cytotoxin. Preferably, D is selected from the group consisting of anthracyclines, camptothecin, meitansinoids, engine, amanitin, auristatin, and pyrrolobenzodiazepine dimers, and more preferably, D is selected from the group consisting of engine, auristatin, and camptothecin. In one embodiment, D is engine. In one embodiment, D is auristatin. In one embodiment, D is camptothecin. Most preferably, D is camptothecin.

[0141] In a preferred embodiment, the engine is selected from calicheamicin, esperamycin, shisijimicin, 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 from the group consisting of topotecan, siratecan, cositecan, exatecan, exatecan-S, DXd, SN-38, lulutotecan, gimatecan, berotecan, rubitecan, AMDCPT, and G-AMDCPT, more preferably from the group consisting of exatecan or DXd, most preferably from the group consisting of exatecan.

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

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

[0144] Preferred antibody conjugate A preferred antibody conjugate according to the first embodiment is selected from the group consisting of compounds (I) to (III), more preferably (II) or (III), and 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 a further preferred embodiment, the antibody conjugate is selected from (XI) and (XIII), more preferably (XIb) or (XIIIe), more preferably the antibody conjugate follows (XIII), and 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 This is defined as -(A) above. d -(B) e -(A) f -(C(O)) g - is a linker represented by -L 2 This is Val-Cit or Val-Ala, -L 3 It is a PABC derivative following the structure (L4), -L 4 is, -N-(C z -alkylene)-C(O)- or -NR 22 -(C z -Alkilen)-NR 22 - and in the formula, z and R 22 This is as defined above, -q = 0 or 1.

[0146] In the context of antibody conjugates (I), L 1 For this, d=1 (A follows structure (23), a=1 and R 13 It is preferable that =H, e=2, f=0 and g=1. In the context of antibody conjugate (I), L 2 =Val-Cit is preferable. In the context of antibody conjugate (I), L 3 Regarding R 21 It is preferable that =H. In the context of antibody conjugate (I), if q=1, it is preferable that z=1 or 5.

[0147] In a particularly preferred embodiment, the antibody conjugate according to structure (I) comprises 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, 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 This is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 This is Val-Cit or Val-Ala, -L 3 It is a PABC derivative following the structure (L4), where R 21 = H.

[0149] In the context of antibody conjugates (II), L follows structure (23). 1 For e=2 and A, a=1 and R 13 It is preferable that =H. In the context of antibody conjugate (II), L 2 It is preferable that =Val-Cit

[0150] In a particularly preferred embodiment, the antibody conjugate according to structure (II) comprises 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, 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) Here, -AB, L 6 Z, D, x, and y are as defined above. -L 1 This is defined as -(A)-(B) above. e A linker represented by -(C(O))-, -L 2 This is Val-Cit or Val-Ala, -L 3 It is a PABC derivative following the structure (L4), where R 21 =H, -L 4 -NR 22 -(C z -Alkilen)-NR 22 - and in the formula, R 22 As defined above, z is an integer between 1 and 6.

[0152] In the context of antibody conjugates (III), L 1 For this, e=2, a=1 and R 13 It is preferable that =H. In the context of antibody conjugate (III), L 2It is preferable that it is Val-Cit. In the context of antibody conjugate (III), it is preferable that z=2.

[0153] In a particularly preferred embodiment, the antibody conjugate according to structure (III) comprises 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, most preferably exatecan as payload D.

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

[0155] L 2 It may be an existence of non-existence, preferably L 2 It exists, and o=1. For the preferred antibody conjugate (Xa), L 2 According to the structure (L3), R 17 It is CH3. For the preferred antibody conjugate (Xb), see L 2 According to the structure (L3), R 17 The compound is CH2CH2CH2NHC(O)NH2. The antibody conjugate (X) preferably has structure (Xa).

[0156] In a particularly preferred embodiment, the antibody conjugate according to structure (X) comprises 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, most preferably exatecan as payload D.

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

[0158] L 2 It may be an existence of non-existence, preferably L 2 It exists, and o=1. For the preferred antibody conjugate (XIa), L 2 According to the structure (L3), R 17 It is CH3. For the preferred antibody conjugate (XIb), see L 2 According to the structure (L3), R 17 The compound is CH2CH2CH2NHC(O)NH2. The antibody conjugate (XI) preferably has structure (XIb).

[0159] In a particularly preferred embodiment, the antibody conjugate according to structure (XI) comprises 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 calicheamicin as payload D.

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

[0161] L 2 It may be an existence of non-existence, preferably L 2 It exists, and o=1. For the preferred antibody conjugate (XIIa), L 2 According to the structure (L3), R 17 It is CH3. For the preferred antibody conjugate (XIIb), see L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2

[0162] L 4 It may be a non-existent presence. For the preferred antibody conjugate (XIIc), q=0, and L 4 It does not exist. For the preferred antibody conjugate (XIId), q=1, and L 4 is structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 20, and R 22 It is either H or a C1-C4 alkyl group.

[0163] For preferred antibody conjugates (XIIe), L 2 According to the structure (L3), R 17 It is CH3, q=0, L 4 It does not exist. For preferred antibody conjugates (XIIf), see L2 According to the structure (L3), R 17 It is CH3, q=1, L 4 Structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 10, preferably z=2, R 22 It is either H or a C1-C4 alkyl group.

[0164] For the preferred antibody conjugate (XIIg), L 2 According to the structure (L3), R 17 It is CH2CH2CH2NHC(O)NH2, and q=0, L 4 It does not exist. For preferred antibody conjugates (XIIh), see L 2 According to the structure (L3), R 17 It is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 20, and R 22 It is either H or a C1-C4 alkyl group.

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

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

[0167] In a particularly preferred embodiment, the antibody conjugate according to structure (XII) comprises 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, most preferably exatecan as payload D.

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

[0169] L 2 It may be an existence of non-existence, preferably L 2 It exists, and o=1. For the preferred antibody conjugate (XIIIa), L 2 According to the structure (L3), R 17 It is CH3. For the preferred antibody conjugate (XIIIb), see L 2 According to the structure (L3), R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH3

[0170] L 4 It may be a non-existent presence. For the preferred antibody conjugate (XIIIc), q=0, and L 4 It does not exist. For the preferred antibody conjugate (XIIId), q=1, and L 4 is structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 20, and R22 It is either H or a C1-C4 alkyl group.

[0171] For preferred antibody conjugates (XIIIe), L 2 According to the structure (L3), R 17 It is CH3, q=0, L 4 It does not exist. For preferred antibody conjugates (XIIIf), see L 2 According to the structure (L3), R 17 It is CH3, q=1, L 4 Structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 10, preferably z=2, R 22 It is either H or a C1-C4 alkyl group.

[0172] For preferred antibody conjugates (XIIIg), L 2 According to the structure (L3), R 17 It is CH2CH2CH2NHC(O)NH2, and q=0, L 4 It does not exist. For preferred antibody conjugates (XIIIh), see L 2 According to the structure (L3), R 17 It is CH2CH2CH2NHC(O)NH2, q=1, L 4 Structure-NR 22 -(C z -Alkilen)-NR 22 - is a diamine spacer according to the formula, where z is an integer in the range of 1 to 20, and R 22 It is either H or a C1-C4 alkyl group.

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

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

[0175] In a particularly preferred embodiment, the antibody conjugate according to structure (XIII) comprises 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, most preferably exatecan as payload D.

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

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

[0178] These preferred antibody conjugates are more preferably conjugated via a glycan (i.e., b=1), more preferably a trimmed glycan (i.e., j=0). In this specification, S=GalNAc and w'=0 are even more preferred. In this specification, the conjugate Z is more preferably formed by azide-alkyne cyclization, preferably by the conjugate Z=(Z39), with ring Z=(Za) and V=CH2. In this specification, x=1 is even more preferred. In this specification, y=2, more preferably x=1 and y=2 are even more preferred.

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

[0180] Compounds following general structure (2) The compound has a general structure (2), QLD (2) During the ceremony, -Q is the reactive part, -L is a linker that connects Z to D, -D is selected from the group consisting of anthracyclines, camptothecin, tubulisin, engine, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, BCL-XL inhibitors, hemiasterin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs.

[0181] Compounds of general structure (2) may also be referred to as “linker-drug constructs” for containing the final conjugate linker L and payload D. Compounds of general formula (2) can 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 a conjugate according to structure (1).

[0182] Reactive part Q Compounds conforming to general structure (2) include a reactive moiety Q. In the context of the present invention, the term “reactive moiety” may refer to a chemical moiety containing a reactive group, but may also refer to the reactive group itself. For example, the cyclooctinyl group is a reactive group, i.e., a reactive group containing a CC triple bond. Similarly, the N-maleimidyl group is a reactive group containing a CC double bond as a reactive group. However, reactive groups, such as azide reactive groups, thiol reactive groups, or alkynyl reactive groups, may also be referred to as reactive moieties in this specification.

[0183] Q is S(F) x It functions as a chemical handle for connection to F. In other words, Q is reactive with F and complementary to F. In this specification, a reactive group is indicated as "complementary" to a reactive group if the reactive group reacts selectively, and optionally, with other functional groups. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Therefore, compounds conforming to general structure (2) are conveniently used in conjugate reactions in which a chemical reaction occurs between Q and F, thereby forming an antibody conjugate involving a covalent connection between payload D and the antibody.

[0184] The exact properties of Q and F depend on the type of conjugation reaction used. Those skilled in the art will be able to select a suitable combination of Q and F. Preferably, Q, and therefore F, are reactive in cycloaddition or nucleophilic reactions. 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 cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, cydonones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or consists of an alkene moiety or an alkyne moiety; more preferably, the alkene is a (hetero)cycloalkene, and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, arenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, vinylsulfones, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety includes or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters and other activated esters, p-nitrophenyl carbonates and other activated carbonates, isocyanates, isothiocyanates, haloacetamides, and benzyl halides. In preferred embodiments, Q is selected from alkene moieties, alkyne moieties, thiol-reactive moieties, or amine-reactive moieties, more preferably alkene moieties or alkyne moieties, and even more preferably alkyne moieties. In this specification, alkenes are preferably (hetero)cycloalkenes, and alkynes are preferably terminal alkynes or (hetero)cycloalkynes. Most preferably, Q is a cyclic (hetero)alkyne moiety. Each of these moieties is further defined below.

[0185] Therefore, in a particularly preferred embodiment, Q includes 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)cyclooctinyl group, a (hetero)cyclononinyl group or a (hetero)cyclodecynyl group. In this specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is optionally substituted with a (hetero)cycloheptynyl group or optionally substituted with a (hetero)cyclooctinyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctinyl group, and the (hetero)cyclooctinyl group is optionally substituted.

[0186] In a particularly preferred embodiment, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and follows structure (Q1). [ka] Here, The bond shown as ---- is a double bond or a triple bond, -R 15 These are, independently, hydrogen, halogen, -OR 16 NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15These are linked together to form an optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero) substituent, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -Y 2 C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 , or NR 31 And in the formula, S (+) B (-) A cationic sulfur atom that is canceled out by, in the formula, B (-) is an anion, and each R 31 R 15 Alternatively, it is 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 within the range of 0 to 16.

[0187] Typically, v = (u + u') × 2 (the connection to L shown by the wave-like combination is Y) 2 (when the connection is via a ripple) or [(u+u')×2]-1 (when the connection to L shown by the ripple 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 follows structure (Q1a). [ka] Here, -R 15 and Y2 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 within the range of 8 to 16.

[0189] In a preferred embodiment, u+u'=4, 5, or 6, and more preferably u+u'=5. In a preferred embodiment, 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 by the wave-like bond, may be a connection 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 functionalized at the option of choice. (-) is an anion, preferably, (-) OTf, Cl (-) , Br (-) , or I (-) Selected from, most preferably B (-) teeth (-) OTf. In the conjugation reaction, B (-) In any case, it exchanges with anions present in the reaction mixture, (-) Q does not need to be a pharmaceutically acceptable anion. When (Q19) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable when the conjugate is isolated according to the present invention, and as a result the conjugate is readily usable 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 the structure (Q38), B (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) or I (-) Selected from, most preferably B (-) teeth (-) It is OTf.

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

[0195] Therefore, in a preferred embodiment, Q includes a heterocycloheptine moiety according to structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptine moiety according to structure (Q37) is not substituted.

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

[0197] In alternative preferred embodiments, Q comprises a (hetero)cyclooctin moiety according to the structures (Q26), (Q27), or (Q28), also referred to as a DIBO, DIBAC, DBCO, or ADIBO group, which are optionally substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctin moiety according to the structures (Q40) or (Q41) shown below, where Y 1 is O or NR 11 And in the formula, R 11 These are, independently, hydrogen, straight-chain or branched-chain C1-C13 12 Alkyl alkyl groups, or C4-C 12 Selected from the group consisting of (hetero)aryl groups. The aromatic ring in (Q40) is optionally O-sulfonylated at one or more positions, while the ring in (Q41) can 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 follows structure (Q43), which is further defined below.

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

[0199] In a particularly preferred embodiment, Q comprises a cyclooctinyl group and follows the structure (Q42). [ka] Here, -R 15 These are, independently, hydrogen, halogen, -OR 16 NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These are linked together to form an optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero) substituent, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -R 18 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups, and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, -R 19These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7-C 24 Alkyl (hetero)aryl groups, and C7-C 24 A group consisting of (hetero)arylalkyl groups is selected, and the alkyl group is optionally interrupted by one of several 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 This is a second appearance of Q or D connected via the spacer portion. -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 These are, independently, hydrogen, halogen, -OR 16 , selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, where R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 These are independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 These are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably both R 18 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 is H. In a preferred embodiment of the reactive group according to structure (Q42), l is 0 or 1, and more preferably l is 1.

[0201] In a particularly preferred embodiment, Q comprises a (hetero)cyclooctinyl group and follows the structure (Q43). [ka] Here, -R 15 is independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1-C 24 alkyl group, C5-C 24 (hetero)aryl group, C7-C 24 alkyl(hetero)aryl group, and C7-C 24 (hetero)arylalkyl group selected from the group consisting of, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (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, and R 16 is independently hydrogen, halogen, C1-C 24 alkyl group, C6-C 24 (hetero)aryl group, C7-C 24 alkyl(hetero)aryl group, and C7-C 24 (hetero)arylalkyl group selected from the group consisting of, -Y is N or CR 15 is.

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

[0203] In a particularly preferred embodiment, Q comprises a heterocycloheptynyl group and follows 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, and 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 a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctenyl group, and the (hetero)cyclopropenyl group, trans-(hetero)cycloheptenyl group, or trans-(hetero)cyclooctenyl group may be optionally substituted. Preferably, Q includes a cyclopropenyl moiety according to structure (Q44), a heteroocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). Here, Y 3 C(R 23 )2, NR 23 , or O is selected, and each R 23The bonds are individually hydrogen, C1-C6 alkyl, or optionally connected to L via spacers, and the bonds labeled --- are single or double bonds. In a further preferred embodiment, the cyclopropenyl group follows structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group follows structure (Q50) or (Q51). In yet another preferred embodiment, the trans-(hetero)cyclooctene group follows 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 suitable for cysteine ​​conjugation. Such probes are known in the art and may be selected from the group consisting of maleimide moieties, haloacetamide moieties, arenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, vinylsulfone, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, Q contains or is a maleimide moiety. The reagent may be monoalkylated or may be a crosslinking agent 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] Here, -X 6is H, halogen, PhS, MeS, preferably halogen such as Cl, Br, I, etc., -X 7 is halogen, PhS, MeS, preferably halogen such as Cl, Br, I, etc., -R 24 is H or C 1~12 alkyl, preferably H or C 1~6 alkyl, -R 25 is H, C 1~12 alkyl, C 1~12 aryl, C 1~12 alkaryl, or C 1~12 aralkyl, preferably H or paramethylphenyl, -where the aromatic rings of (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.

Chemical formula

[0209] In an alternative, preferred embodiment, Q is an amine-reactive probe. In this embodiment, Q is a reactive group suitable for a 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 a 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] Here, X 2 This is a halogen, preferably F.

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

[0212] Antibodies following the general structure (3) Antibodies have a general structure (3), AB-[(L 6 ) b -{F} x ] y (3) During the ceremony, -AB is an antibody that can target PTK7-expressing tumors. -b is either 0 or 1. -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - is a monosaccharide, where G is a monosaccharide, j is an integer in the range of 0 to 10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, L7 These are -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. -F is the reactive part, -x is either 1 or 2, -y is 1, 2, 3, or 4.

[0213] The antibody of general structure (3) may also be called a "(modified) antibody" because it contains a reactive group F, which is either naturally occurring or the antibody is modified to incorporate the reactive group F. A (modified) antibody according to general formula (2) can be prepared by those skilled in the art using standard organic and / or enzymatic synthesis techniques, as illustrated in the examples. Antibody AB, linker L 6 b, x, and y are defined above in the context of a conjugate following structure (1).

[0214] Reactive part F F is reactive with Q in a conjugation reaction as defined below, preferably the conjugation reaction is a cycloaddition or nucleophilic reaction. As those skilled in the art will understand, the choices for F are the same as the choices for Q, except that F and Q are reactive with each other. Therefore, 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 cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, cydonones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is one of the following: azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, or cydonones, most preferably azides. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, arenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, orthoquinone moieties, vinylsulfones, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety includes or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters, isocyanates, isothiocyanates, and benzyl halides. In preferred embodiments, F is a click probe or a thiol, more preferably F is an azide or a thiol, and most preferably F is an azide.

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

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

[0217] Here, a wave coupling represents a connection to the payload. For (F3), (F4), (F8), and (F9), the payload can be connected to any one of the wave couplings. The other wave couplings then connect to hydrogen, C1-C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (Hetero)arylalkyl groups, and C1-C 24 R groups selected from sulfonyl groups may be attached, and each (except hydrogen) may be optionally substituted with O, S, and NR. 32 It may be interrupted by one or more heteroatoms selected from R32 The R group is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Those skilled in the art will understand which R group can be applied to each of the groups F. For example, the R group linked to the nitrogen atom of (F3) may be selected from alkyl and aryl groups, and the R group linked to the carbon atom of (F3) may be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl groups. Preferably, the reactive moiety F is selected from azide or tetrazine. Most preferably, the reactive moiety F is an azide.

[0218] In a second preferred embodiment, F is a thiol or its precursor. The thiol or its precursor F is used in a conjugation reaction to link the linker-drug construct to the (modified) antibody. F is reactive to the thiol-reactive probe Q in thiol ligation. The thiol is preferably a thiol of the side chain of a naturally occurring cysteine ​​amino acid in antibody AB, in which case the linker L 6 It does not exist (b=0), but linker L can be chosen at will. 6 It may be introduced synthetically via [a specific method]. The thiol precursor in the context of bioconjugation includes a disulfide that is known in the art and present in antibodies, which may be a naturally occurring disulfide bridged disulfide, or a synthetically introduced disulfide that is 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 its precursor, preferably an amine. The amine or its precursor F is used in a conjugation reaction to link the linker-drug construct to the (modified) antibody. F is reactive to the amine-reactive probe Q in nucleophilic substitution. The amine is typically a primary amine, preferably an amine of the side chain of a naturally occurring lysine amino acid in antibody AB, in which case the linker L 6 It does not exist (b=0), but linker L can be chosen at will. 6It may also be introduced synthetically via [a specific method]. Preferably, F is a primary amine group in the lysine side chain.

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

[0221] The multifunctional antibody constructs according to the present invention can be prepared using any conjugation technique known in the art. Preferred conjugation techniques include thiol ligation, lysine ligation, and cyclization (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cyclization, strain-promoted quinone-alkyne cyclization). Preferred conjugation techniques used in the context of the present invention include nucleophilic reactions and cyclization, preferably the cyclization being [4+2] or [3+2], and the nucleophilic reaction being Michael addition or nucleophilic substitution. Suitable conjugation techniques are disclosed, for example, in G. Thermanson, “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] Therefore, in a preferred embodiment of the conjugation process according to the present invention, conjugation is achieved via nucleophilic substitution or a nucleophilic reaction such as a Michael reaction. A preferred nucleophilic reaction is the acylation of a primary amino group by an activated ester. A preferred Michael reaction is the maleimide-thiol reaction, which is widely used in bioconjugation.

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

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

[0225] Therefore, a process for preparing an antibody conjugate according to the present invention in accordance with this embodiment includes reacting an antibody modified to 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 the antibody conjugate according to the present invention is: (i) An antibody containing the y-core N-acetylglucosamine (GlcNAc) moiety (y = 1, 2, 3, or 4) is subjected to formula S(F) in the presence of a catalyst. x -P(in the formula, S(F) x A is a sugar derivative containing x reactive groups F that can react with reactive group Q, where x is 1 or 2, P is a monophosphate or diphosphate nucleoside, and the catalyst is S(F) x The compound (which can be transferred to the core-GlcNAc portion) is brought into contact with the compound of formula (26) AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) (In the formula, -AB is an antibody that can target PTK7-expressing tumors. -Fuc is fucose, -w is 0 or 1) to obtain a modified antibody according to, (ii) Modified antibody, structure (2) QLD (2) (In the formula, -Q is the reactive part, -L is a linker that connects Z to D, -D is reacted with a compound selected from the group consisting of anthracyclines, camptothecin, tubulisin, enediyne, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, BCL-XL inhibitors, hemiasterin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs. This includes obtaining an antibody conjugate that conforms to structure (1).

[0227] Step (i) In step (i), an antibody containing one, two, three, or four core N-acetylglucosamine moieties is subjected to the reaction of formula S(F) in the presence of a catalyst.x -P is brought into contact with the compound, but in the formula, S(F) x P is a sugar derivative containing x reactive groups F that can react with reactive group Q, where x is 1 or 2, P is a nucleoside monophosphate or diphosphate, and the catalyst is S(F) x The portion can be transferred to the core-GlcNAc portion. In this specification, the antibody is typically an antibody trimmed to the core-GlcNAc residue, as further described below. Step (i) provides a modified antibody according to formula (26).

[0228] The starting material, i.e., the antibody containing the core-GlcNAc substituent, is known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the process according to the present invention further comprises deglycosylating an antibody glycan having core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody containing the core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the properties of the glycan, a suitable endoglycosidase may 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, and the selection depends on the properties of the glycan. For EndoSH, please refer to PCT / EP2017 / 052792, which is incorporated herein by reference; see Examples 1-3 and Sequence ID 1.

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

[0230] SCIENCE FICTION) x Suitable catalysts capable of transferring a portion to the core-GlcNAc portion are known in the art. A suitable catalyst is one that can transfer a specific sugar derivative nucleotide S(F) in its particular process. x-P is a substrate catalyst. 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-acetylgalactosaminyltransferase (GalNAcT) and β(1,4)-galactosyltransferase (GalT), and most preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain. Suitable catalysts and their mutations are disclosed in WO2014 / 065661, WO2016 / 022027, and WO2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably N-acetylgalactosaminyltransferase. In alternative embodiments, 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 can recognize internal sugars and sugar derivatives as acceptors. Therefore, sugar derivative S(F) x This is linked to the core GlcNAc substituent in step (i), regardless of whether the GlcNAc is fucosylated or not.

[0231] Step (i) is preferably carried out in a suitable buffer solution such as 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, more preferably in the range of about 5.5 to about 8.5, and 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 conforming to a general structure (2) that contains a reactive group F and a reactive group Q that can react with the payload D, to obtain an antibody conjugate conforming to structure (1) that contains a conjugating group Z resulting from the reaction between Q and F. Such a reaction occurs under conditions such that the reactive group Q reacts with the reactive group F of the biomolecule, covalently bonding the antibody to the compound conforming to general structure (2). Step (ii) may also be called 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 following 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 must 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 may occur spontaneously by a reaction called strain-enhanced azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in this field as "metal-free click chemistry."

[0234] application The present invention relates to a method for treating a subject in need of treatment, comprising the administration of an antibody conjugate according to the present invention as defined above. Subjects in need of treatment are typically cancer patients. The use of antibody conjugates, such as antibody-drug conjugates, is well known in the field of cancer treatment, and antibody conjugates according to the present invention are particularly suitable in this respect. The described method is typically suitable for the treatment of cancer. In this embodiment of the method, the antibody conjugate is typically administered in a therapeutically effective dose. This embodiment of the present invention can also be expressed as an antibody conjugate according to the present invention for use in the treatment of a subject in need of treatment, preferably for use in the treatment of cancer. In other words, this embodiment relates to the use of an antibody conjugate according to the present invention for the preparation of a drug or pharmaceutical composition for use in the treatment of a subject in need of treatment, preferably for use in the treatment of cancer. In this context, cancer treatment is assumed to encompass the treatment, imaging, diagnosis, prevention, inhibition, and reduction of tumor growth.

[0235] This aspect of the present invention may also be expressed as a method for targeting PTK7-expressing cells, particularly PTK7-expressing tumor cells, and comprises contacting an antibody conjugate according to the present invention with cells that may express PTK7. Therefore, the method according to this aspect is suitable for determining whether cells express PTK7. These PTK7-expressing cells may be present in a subject, in which case the method comprises administering the antibody conjugate according to the present invention to the subject requiring it. In a preferred embodiment, cells that may express PTK7 are PTK7-expressing cells. Targeting PTK7-expressing cells preferably includes one or more of the following: treatment, imaging, diagnosis, prevention, inhibition, and reduction of proliferation of PTK7-expressing cells, particularly PTK7-expressing tumor cells. The method according to this embodiment may be medical or non-medical. A non-medical method according to this aspect may target PTK7-expressing cells in vitro or ex vivo, where cells that may express PTK7 are present, for example, in a sample taken from a patient. Such non-medical methods are typically used in the diagnosis of cancer, particularly PTK7-positive cancer.

[0236] In the context of the present invention, subjects may be suffering from a disorder selected from colon cancer, lung cancer, breast cancer, ovarian cancer, and esophageal cancer. Therefore, the treatment of subjects requiring it preferably refers to the treatment of colon cancer, lung cancer, breast cancer, ovarian cancer, and esophageal cancer.

[0237] To our surprise, the inventors have found that the antibody conjugate according to the present invention is superior to conventional PTK7-targeted antibody conjugates in terms of safety and / or efficacy, resulting in an increased therapeutic index of the antibody conjugate according to the present invention.

[0238] Conjugation style In relation to the present invention, “conjugation style” refers to the process used to conjugate payload D to antibody AB, and the resulting antibody conjugate, in particular the structural features of the linker that connects the payload to the antibody, which are the direct result of the conjugation process. Thus, in one embodiment, conjugation style refers to the process for conjugating the payload to the antibody. In an alternative embodiment, conjugation style refers to the structural features of the linker and / or the linker's binding site to the antibody, which are the direct result of the process for conjugating the payload to the antibody.

[0239] In a further embodiment, the present invention relates to the use of a conjugation mode to increase the therapeutic index of an antibody conjugate in the treatment of PTK7-expressing tumors, wherein the conjugation mode is used to connect an antibody AB to a payload D via a linker L. (i) An antibody containing the y-core N-acetylglucosamine (GlcNAc) moiety (y = 1, 2, 3, or 4) is subjected to formula S(F) in the presence of a catalyst. x -P(in the formula, S(F) x A is a sugar derivative containing x reactive groups F that can react with reactive group Q, where x is 1 or 2, P is a monophosphate or diphosphate nucleoside, and the catalyst is S(F) x The compound (which can be transferred to the core-GlcNAc portion) is brought into contact with the compound of formula (26) AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) (In the formula, -AB is an antibody that can target PTK7-expressing tumors. -Fuc is fucose, -w is 0 or 1) to obtain a modified antibody according to, (ii) Modified antibody, structure (2) QLD (2) (In the formula, -Q is the reactive part, -L is a linker that connects Z to D, -D is reacted with a compound selected from the group consisting of anthracyclines, camptothecin, tubulisin, enediyne, amanitin, duocalmycin, meitansinoids, auristatin, eribulin, BCL-XL inhibitors, hemiasterin, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs), and their analogs or prodrugs. This includes obtaining an antibody conjugate that conforms to structure (1).

[0240] Preferably, increasing the therapeutic index of the antibody conjugate is (a) to increase the therapeutic efficacy of antibody conjugates, and / or (b) Selected because it increases the tolerability of the antibody conjugate.

[0241] The increased therapeutic efficacy of the antibody conjugate according to the present invention may take the form of a reduction in tumor size and / or an extension of the regression period compared to conventional PTK7-targeted ADCs. The increased tolerability of the antibody conjugate according to the present invention may take the form of a reduction in signs of toxicity compared to the administration of PTK7-targeted ADCs produced by conventional techniques. This reduction in signs may also be referred to as a reduction in symptoms or side effects of cancer treatment and may be accompanied by one or more clinical signs such as reduced weight loss, reduced mobility, reduced food intake, and / or improvements in one or more toxic parameters such as 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 in 1 L, 100 mL, 100 mL, and 5 L scales by Evitria (Zurich, Switzerland). Other IgGs (12C6 and 12C6a) were transiently expressed in ExpiCHO-S(TM) cells (Gibco) in 500 mL scales. Non-fucosylated 12C6 was transiently expressed in CHO cells using ProBioGen's GlymaxX® technology by Evitria. The supernatant was purified using a HiTrap MabSelect sure column. The supernatant was loaded into 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. Next, IgG was concentrated using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) (>20 mg / mL). The IgG sequence 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 (Sequence ID 16):

number

[0246] 4D5(II) heavy chain (SEQ ID NO: 15):

number

[0247] 7C8(III) light chain (Sequence ID 25):

number

[0248] 7C8(III) heavy chain (SEQ ID NO: 26):

number

[0249] 12C6(IV) light chain (Sequence ID 20):

number

[0250] 12C6(IV) heavy chain (SEQ ID NO: 19):

number

[0251] 12C6a(V) light chain (Sequence ID 22):

number

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

[0253] B12(VI) Light Chain (Sequence ID 28):

number

[0254] B12(VI) heavy chain (SEQ ID NO: 27):

number

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

[0256] General procedure for mass spectral analysis of (modified) monoclonal antibodies: Prior to mass spectrometry, IgG was treated with IdeS to enable analysis of the Fc / 2 fragment. For analysis of both light and heavy chains, a solution of 20 μg of (modified) IgG was incubated with 100 mM DTT in a total volume of 4 μL at 37°C for 5 minutes. If present, azide functional groups are reduced to amines under these conditions. For analysis of the Fc / 2 fragment, a solution of 20 μg of (modified) IgG was incubated with IdeS / Fabricator™ (1.25 U / μL) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL at 37°C for 1 hour. After diluting the sample to 80 μL, the electrospray ionization time of flight (ESI-TOF) was analyzed on a JEOL AccuTOF. Inverse superimposed 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 at 30°C for 16 hours with 1% w / w EndoSH (see Examples 1-3 and SEQ ID NO: 1, as described in PCT / EP2017 / 052792 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 incorporated herein by reference), 6 mM MnCl2, and 0.01% AP(Roche) and UDP6-N3-GalNAc (compound 2d in Figure 3, 25 equivalents compared to IgG) in TBS. The functionalized IgG was then purified using a HiTrap MabSelect sure 5 mL column. After adding 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 cycles to 20 mM histidine and 150 mM NaCl pH 7.5, the 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 standard procedure for enzyme remodeling. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the expected product (observed mass 24329 Da, approximately 90% of total Fc / 2).

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

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

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

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

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

[0264] Example 7: Preparation of aFuc-12C6-(6-N3-GalNAc)2(aFuc-IV-N3) Following a standard procedure for enzyme 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 corresponding to the expected product (observed mass 24187 Da, approximately 95% of total Fc / 2).

[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 hours, the reaction mixture was diluted with 9 mL of DCM and purified by gradient column chromatography (0 → 40% MeOH / DCM) to afford 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 FN6O 10 + (M+H) + Calculated value for 977.39, found 977.72. In addition to 11, free base of exatecan (82.4 mg, 189 μmol, 20%) was recovered. LCMS (ESI+) C 24 H 23 FN3O4 + (M+H) + Calculated value for 436.46, found 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. 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 hours, diluted with DCM (20 mL) and purified by gradient column chromatography (0 → 40% MeOH / DCM) to afford 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)<0C000984>Calculated value for 1066.88, found 1067.12. <OO02405>

Chemical Structure

[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), triethylamine (22 μL, 16 mg, 158 μmol) and a solution of vc-PABC-MMAE.TFA13 (96 mg, 78 μmol) in DMF (1.0 mL) were added. The mixture was allowed to stand for 19 hours, and 2,2'-(ethylenedioxy)bis(ethylamine) (37 μL, 38 mg, 253 μmol) was added. After 2 hours, 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%). LC-MS (ESI + )C 138 H 219 N 23 O 35 S 2+ (M+2H + The calculated value was 1395.79, and the measured value was 1396.31.

[0268] Example 11: Synthesis of linker conjugate 5b The synthesis of BCN-HS-vc-PABC-CM(5b) was carried out 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), 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 / vEt3N in DMF (8.98 μL, 32.2 μmol) were added. The mixture was allowed to stand for 4 hours and then diluted to a total volume of 4.5 mL with DCM. This 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 + The calculated value was 1115.35, and the measured value was 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 equivalent) 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 equivalents) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 equivalents), and HATU (8.9 mg, 23 μmol, 2.3 equivalents). After stirring at room temperature for 2 hours, 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 obtain the product as a clear oil (determining the yield was difficult due to the presence of DMF). LCMS(ESI+)C 140 H 150 F2N 17 O 33 S + (M / 2+H + The calculated value was 1334.01, and the measured value was 1334.79.

[0270] This compound was dissolved in DMF (300 μL), and triethylamine (21 μL, 150 μmol, 15 equivalents) was added. After 17 hours at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by flash column chromatography through silica gel (0% → 45% MeOH in DCM) to obtain 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 +The calculated value was 1156.2, and the measured value was 1156.74.

[0271] Examples 13-24: Conjugation of linker payload to (modified) monoclonal antibody Example 13: Preparation of cofetuzumab-Aur0101 Cofetuzumab (14.4 mg, 4.34 mg / ml in 20 mM histidine, 6% sucrose) was mixed with 5% pH-adjusted buffer (0.5 M Tris, 25 mM EDTA, pH 8.5) and 2.3 molar equivalents of TCEP (1 mM in water), and incubated at room temperature for 2 hours. DMA was added to the reduced antibody to obtain 10% v / v and 6 molar equivalents of auristatin 0101 (2 mM in DMA), and incubated at room temperature for 1 hour. The reaction was quenched using 6 molar equivalents of NAC (100 mM in water). The conjugate was purified by preparative discontinuous diafiltration in Vivaspin into 20 mM histidine / 6% sucrose. 0.04% Tween-20 was added before filter sterilization. The mean DAR was measured and was 4.3.

[0272] Example 14: Conjugation of cofetuzumab (6-N3-GalNAc)2 and 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), sodium deoxycholate (322 μL, 110 mM), BCN-HS-PEG2-HS-(va-PAB-Ex)23 (86 μL, 10 mM solution in DMF), and propylene glycol (881 μL) were added. The reaction mixture was incubated overnight at room temperature. The conjugate was then purified on a HiLoad 16 / 600 Superdex200 PG column (Cytiva) on AKTA Pure (Cytiva). To remove excess free payload, 25 mg of activated carbon (Carbon RHC, Filtrox AG) was added and the mixture was rotated overnight. The char was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, Corning). Next, the solution was buffer-exchanged using a HiTrap26-10 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 filtered to sterilize. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to conjugate cofetuzumab-3 (observed mass 26461 Da, approximately 90% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean 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), BCN-HS-(vc-PABC-MMAE)24 (1.8 μL, 100 mM solution in DMF), and DMF (58 μL) were added. The reaction mixture was incubated overnight at room temperature. The conjugate was then purified on a HiLoad 10 / 300 Superdex200 PG column (Cytiva) on AKTA Pure (Cytiva). Next, the solution was buffer-exchanged using a HiTrap26-10 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 filtered to sterilize. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to conjugate cofetuzumab-4 (observed mass 27119 Da, approximately 90% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean 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), sodium deoxycholate (94 μL, 110 mM), BCN-HS-vc-PABC-calicheamicin 5b (7 μL, 40 mM solution in DMF), and DMF (87 μL) were added. The reaction mixture was incubated overnight at room temperature. Next, the conjugate was purified on a HiLoad 10 / 300 Superdex200 PG column (Cytiva) on AKTA Pure (Cytiva). Subsequently, the solution was buffer-exchanged using a HiTrap26-10 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 filtered to sterilize. Mass spectral analysis of the sample after IdeS treatment revealed one major Fc / 2 product corresponding to conjugate cofetuzumab-5b (observed mass 26714 Da, approximately 90% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 1.93.

[0275] Example 17: Conjugation of B12(6-N3-GalNAc)2 and 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), BCN-HS-PEG2-HS-(va-PAB-Ex)23 (495 μL, 10 mM solution in DMF) and propylene glycol (7.0 mL) were added. The reaction mixture was incubated overnight at room temperature. Next, the conjugate was purified on a HiLoad 26 / 600 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva) system. To remove excess free payload, 150 mg of activated carbon (Carbon RHC, Filtrox AG) was added and the mixture was rotated overnight. The char was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, Corning). Next, the solution was dialyzed to 20 mM histidine, 6% sucrose buffer, pH 6.0, at room temperature for 2 hours and overnight at 4°C. The solution was concentrated using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius), 0.04% Tween-20 was added, and the solution was filtered and sterilized. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to conjugate B12-3 (observed mass 26462 Da, approximately 90% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.75.

[0276] Example 18: Conjugation of 4D5(6-N3-GalNAc)2 and 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), sodium deoxycholate (88 μL, 110 mM), BCN-HS-PEG2-HS-(va-PAB-Ex)23 (23.4 μL, 10 mM solution in DMF), and propylene glycol (240 μL) were added. The reaction mixture 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 on AKTA Pure (Cytiva) with PBS pH 7.4. To remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added, and the mixture was rotated overnight. The charcoal was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, Corning). The solution was then dialyzed three times, and 20 mM histidine, 6% sucrose buffer (pH 6.0), and 0.04% Tween-20 were added before sterilization by filtration. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to conjugate 4D5-3 (observed mass 26465 Da, approximately 90% of total Fc / 2), as well as one small amount of Fc / 2 product corresponding to the non-fucosylated product (observed mass 26318 Da, approximately 10% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.91.

[0277] Example 19: Conjugation of 7C8(6-N3-GalNAc)2 and 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), BCN-HS-PEG2-HS-(va-PAB-Ex)23 (23.1 μL, 10 mM solution in DMF) and propylene glycol (254 μL) were added. The reaction mixture was incubated overnight at room temperature. The conjugate was then purified on a Superdex200 10 / 300 increasing column (Cytiva) on AKTA Pure (Cytiva). To remove excess free payload, 11 mg of activated carbon (Carbon RHC, Filtrox AG) was added and the mixture was rotated overnight. The carbon was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, Corning). Next, the solution was 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 filtered to sterilize. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the conjugate 7C8-3 (observed mass 26466 Da, approximately 90% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.95.

[0278] Example 20: Conjugation of 12C6(6-N3-GalNAc)2 and BCN-HS-PEG2-HS-(va-PAB-Ex)23 to obtain conjugate 12C6-3 A solution of 12C6(6-N3-GalNAc)2 (481 μL, 10.1 mg, 21.01 mg / ml in TBS pH 7.5) was mixed with BCN-HS-PEG2-HS-(va-PAB-Ex)23 (33.7 μL, 10 mM solution in DMF) and propylene glycol (370 μL). The reaction mixture 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 on AKTA Pure (Cytiva) with PBS pH 7.4. To remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added and the mixture was rotated overnight. The charcoal was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, Corning). Next, the solution was 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 filtered and sterilized. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the conjugate 12C6-3 (observed mass 26466 Da, approximately 95% of total Fc / 2), as well as one small amount of Fc / 2 product corresponding to the non-fucosylated product (observed mass 26318 Da, approximately 5% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.85.

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

[0280] Example 22: Conjugation of 12C6(6-N3-GalNAc)2 and 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), sodium deoxycholate (100 μL, 110 mM), BCN-HS-PEG2-(eva-PAB-Ex)29 (20 μL, 10 mM solution in DMF), and propylene glycol (280 μL) were added. The reaction mixture 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 on AKTA Pure (Cytiva) with PBS pH 7.4. To remove excess free payload, 12 mg of activated carbon (Carbon RHC, Filtrox AG) was added, and the mixture was rotated overnight. The charcoal was removed by centrifugation, followed by filtration through a PES syringe filter (0.20 μm pore, 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 filtered to sterilize. Mass spectrometry of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the conjugate 12C6-9 (observed mass 26466 Da, approximately 95% of total Fc / 2), as well as one small amount of Fc / 2 product corresponding to the non-fucosylated product (observed mass 26643 Da, approximately 5% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.84.

[0281] Example 23: Conjugation of aFuc-12C6(6-N3-GalNAc)2 and 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), sodium deoxycholate (130 μL, 110 mM), BCN-HS-PEG2-HS-(va-PAB-Ex)23 (34.7 μL, 10 mM solution in DMF), and propylene glycol (355 μL) were added. The reaction mixture was incubated overnight at room temperature. Next, the conjugate was purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, and equilibrated on AKTA Pure (Cytiva) with PBS pH 7.4. Next, the solution was 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 filtered and sterilized. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product corresponding to the conjugate aFuc-12C6-3 (observed mass 26318 Da, approximately 95% of total Fc / 2). RP-UPLC analysis of the sample under reducing conditions showed a mean DAR of 3.74.

[0282] Example 24: Conjugation of aFuc-12C6(6-N3-GalNAc)2 and 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), sodium deoxycholate (14 μL, 110 mM), BCN-HS-PEG2-(eva-PAB-Ex)29 (2.8 μL, 10 mM solution in DMF), and propylene glycol (39 μL) were added. The reaction mixture was incubated overnight at room temperature. Next, the conjugate was purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2 M NaOH, and equilibrated on AKTA Pure (Cytiva) with PBS pH 7.4. RP-UPLC analysis of the conjugate aFuc-12C6-9 sample under reducing conditions showed a mean DAR of 3.81.

[0283] Examples 25-28: In vitro assay Example 25. ELISA assay for the binding of hPTK7, cPTK7, and rPTK7 to mAbs and ADCs. Nickel NTA plates (Pierce® nickel-coated plates, ThermoScientific®) were washed three times before use. Human PTK7 (CCK4 protein, ECD, His Tag, Sino Biological), cynomolgus monkey PTK7 (CCK4 protein, ECD, His Tag, Sino Biological), and rat PTK7 (His Tag, Acro Biosystems) were dissolved in PBS (PBA) at a concentration of 0.05 μg / mL in 0.1% BSA. 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 (wash buffer) in PBS. ADC was diluted in 0.1% PBA to a final concentration of 8 μg / mL and 100 μL was added to each well (triple). The ADC was incubated at room temperature for 1 hour. The plate was washed three times with washing buffer before adding 100 μL of a 1:1000 dilution of the secondary antibody (goat anti-human IgG, HRP conjugate, Invitrogen). The plate was incubated again at room temperature for 1 hour, then washed three times with washing 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. The absorbance of the colorimetric signal was measured at 652 nm using Infinite® M1000 (Tecan). The data were plotted corrected for background (see Figures 8A, 8B and 11).

[0284] Example 26. Human and mouse serum stability The stability of 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 rapidly frozen and stored at -80°C until further analysis. To isolate ADC after incubation, 20 μl of CaptivA® Protein A agarose resin was added to the sample and incubated at room temperature for 1 hour. The resin was washed three times with PBS, followed by elution of ADC with 0.4 mL of 0.1 M glycine-HCl (pH 2.7). After elution, the sample was immediately neutralized with 0.1 mL of 1.0 M Tris pH 8.0. The sample was spin-filtered three times against PBS using an Amicon Ultra spin filter 0.5 mL MWCO 10 kDa (Merck Millipore) to reduce the volume to 40 μL, obtaining a final ADC concentration of approximately 1 mg / mL. The sample was analyzed by RP-UPLC (DTT reduction) to determine the DAR (see table below). [Table 1] [Table 2] -†Signal is too low *Direct measurement (not by separation from plasma using protA agarose resin). The values ​​in parentheses represent the percentage for t=0.

[0285] Example 27: In vitro cytotoxicity A431 (PTK7+, ATCC CRL-1555) cells were seeded in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) into 96-well plates (10,000 cells per 150 μL / well) and incubated overnight at 37°C in a humidified atmosphere of 5% CO2. ADC was added in triple doses using the square root of 10 dilution series to obtain final concentrations ranging from 100 nM to 0.01 nM. The cells were incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2. The culture medium was replaced with 0.01 mg / mL of rezazlin (Sigma Aldrich) in DMEM supplemented with 10% FBS (200 μL / well). After approximately 4 hours in a humidified atmosphere of 37°C and 5% CO2, fluorescence was detected using a fluorescence plate reader (Infinite® M1000 Tecan) with excitation at 560 nm and emission at 590 nm. Relative fluorescence units (RFU) were normalized to the cell viability percentage by setting cell-free wells to 0% viability and wells containing untreated cells to 100% viability (see Figure 9). IC50 of ADCs on A431 cells. 50 The values ​​were calculated using nonlinear regression with Graphpad Prism software and are shown in the table below. [Table 3]

[0286] Example 28. Binding affinity of 12C6-3 and 12C6 naked mAbs to FcγR using Biacore The binding of test antibodies to high-affinity and low-affinity human Fc gamma receptors was evaluated by single-cycle analysis using a Biacore T200 instrument (serial number 1909913) running Biacore T200 evaluation software (Cytiva, Uppsala, Sweden) 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). FcγR was captured on a pre-bound CM5 sensor chip using a His capture kit (Cytiva, Uppsala, Sweden) using standard amine chemistry.

[0287] At the start of each cycle, His-tagged Fc gamma receptors (ligands) diluted with HBS-P+ (Cytiva, Uppsala, Sweden) were loaded to a specific RU level. Five points, a 3-fold dilution range of test antibodies (analytes) without regeneration between concentrations were used for each receptor tested. The test antibodies were passed over the chip at increasing concentrations of 30 μl / min, followed by a single dissociation step. After dissociation, the chip was regenerated by injecting glycine pH 1.5. The signal from reference channel Fc1 (blank) was subtracted from the signal of receptor-loaded Fc to correct for differences in nonspecific binding to the reference surface. Sensorgrams were analyzed by 1:1 dynamics of the high-affinity Fc gamma receptor hFcγRI and by 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 binding scale (KD) is determined by Biacore, from +++++ to 10. -9 ~10 -10 Range of M, ++++, 10 -8 Range of M, +++, 10 -7 Range of M, ++, 10 -6 M, +, 10 -5 This indicates the range of M. [Table 4-1]

[0289] Examples 29-34: CDX evaluation and in vivo studies Example 29: IHC staining of PTK7 with NCI-H446 (performed at Crown Bioscience Taicang, China) Formalin-fixed paraffin-embedded (FFPE) blocks were cut onto glass slides to a thickness of 4 μm on a manual rotary microtome. The slides were calcined at 60°C for 30 minutes. Staining was performed using Bond RX autostainer (Leica) following these steps: The slides were first treated with dewaxing solution (Leica) at 72°C for 0.5 minutes, then treated at room temperature, transferred to alcohol, and treated three times at room temperature. Next, the slides were washed three times with Bond Wash buffer (Leica). After rinsing quickly in the same solution, epitope recovery was performed at 100°C in Bond ER2 solution (Leica) for 20 minutes. Subsequently, the slides were washed four times for short periods in Bond wash buffer, followed by washing at room temperature for 3 minutes. The peroxide blocks were treated at room temperature for 10 minutes, and then washed three more times. Subsequently, the primary antibody rabbit anti-human PTK7 (PA5-82070, Thermo Fisher, 0.25 μg / mL) was added and incubated at room temperature for 1 hour. Subsequently, a single rapid wash and three 2-minute washes were performed with Bond wash buffer. The secondary antibody, goat anti-rabbit HRP (Leica, ready to use), was added and incubated at room temperature for 20 minutes, followed by a single rapid wash and three 2-minute washes in Bond wash buffer. After rinsing the slides with deionized water, they were incubated with 3,3'-diaminobenzidine (DAB)Refine at room temperature for 5 minutes. After three washes with deionized water, the slides were treated with hematoxylin as a control dye for 10 minutes. Finally, all slides were washed with deionized water and binding wash buffer.

[0290] All stained sections were scanned at 40x magnification using the NanoZoomer-HT 2.0 imaging system. All images of non-necrotizing tumor areas were analyzed on the HALO® platform. The intensity of membrane-positive staining was scored on a four-level scale: 0 (negative), 1+ (weak staining), 2+ (medium staining), and 3+ (strong staining). The percentage of cells at different intensity levels was evaluated using the H-score. H-score = (%) at 0 × 0 + (%) at 1 × 1 + (%) at 2 × 2 + (%) at 3 × 3 (H-score range: 0 to 300). For the H-score of the NCI-H446 lung cancer xenograft cell line, please refer to Table 4. [Table 4-2]

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

[0292] 7-9 week old BALB / c nude female mice were given 5 × 10 in 0.1 ml of PBS mixed with Matrigel (1:1) to induce tumor formation. 6 Individual tumor cells were subcutaneously injected into the right anterior flank region. The tumor was 100-200 mm in size. 3 Once the mice reached their average size, they were randomized into 10 groups of 8 mice each, and treatment was initiated. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0293] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and the animals' body weight was measured twice a week. The evaluation item for the experiment was 3,000 mm 3This is defined as the tumor volume, a weight loss of more than 20%, or weight loss over 28 days, whichever comes first.

[0294] Randomization: Average tumor size is approximately 100-200 mm 3 Randomization was performed once the target number of mice was reached. A total of 80 mice were enrolled in the NCI-H446 model study and randomly assigned to 10 groups of 8 mice each. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0295] Administration of test sample: The test sample was administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment was initiated on the same day as randomization. The medication was administered in a laminar flow cabinet.

[0296] Observation and data collection. After inoculation of tumor cells, the morbidity and mortality rates of the animals are checked daily. At the point of routine monitoring, the animals are checked for adverse effects of tumor growth and treatment on normal behavior, such as mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair matting, and any other abnormal effects. Tumor volume is measured in two dimensions every 3-4 days using calipers, and the volume data is expressed in mm³ using the formula V=(L×W×W) / 2, 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). Medication, as well as tumor and weight measurements, are performed in a laminar flow cabinet. [Table 5]

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

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

[0299] 7-9 week old BALB / c nude female mice were given 5 × 10 in 0.1 ml of PBS mixed with Matrigel (1:1) to induce tumor formation. 6 Individual tumor cells were subcutaneously injected into the right anterior flank region. The tumor was 100-200 mm in size. 3 Once the mice reached their average size, they were randomized into six groups of eight mice each, and treatment was initiated. Randomization was performed using the "matched distribution" method (Study Director™ software, version 3.1.399.19). The randomization date is indicated as day 0.

[0300] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and the animals' body weight was measured twice a week. The evaluation item for the experiment was 3,000 mm 3 This is defined as tumor volume, a weight loss of more than 20%, or weight loss over 77 days, whichever comes first.

[0301] Randomization: Average tumor size approximately 136 mm 3 Randomization was performed once the target number of mice was reached. A total of 48 mice were enrolled in the NCI-H446 model study and randomly assigned to 6 groups of 8 mice each. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0302] Administration of test sample: The test sample was administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment was initiated on the same day as randomization. The medication was administered in a laminar flow cabinet.

[0303] Observation and Data Collection: After inoculation with tumor cells, the morbidity and mortality rates of the animals are checked daily. At the point of routine monitoring, the animals are checked for adverse effects of tumor growth and treatment on normal behavior, including mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair matting, and any other abnormal effects. Tumor volume is measured two-dimensionally every 3-4 days using calipers, and the volume data is expressed in mm³ using the formula V=(L×W×W) / 2, 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). Medication, as well as tumor and weight measurements, are performed in a laminar flow cabinet. [Table 6]

[0304] The tumor volume and mouse body weight over time in the efficacy study, as well as the corresponding Kaplan-Meier plots using the above-mentioned test items, are shown in Figures 12A, 12B, and 12C.

[0305] Example 32: In vivo efficacy study 7-9 week old BALB / c nude female mice were loaded with tumor fragments from BR1282, a xenograft model cell line derived from breast cancer patients. Tumor fragments (2-3 mm in diameter) were subcutaneously inoculated into the right upper flank region. The tumors grew to 100-200 mm. 3 Once the mice reached their average size, they were randomized into nine groups of eight, and treatment was initiated. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0306] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and the animals' body weight was measured twice a week. The evaluation item for the experiment was 3,000 mm 3 This is defined as the tumor volume, a weight loss of more than 20%, or weight loss over 39 days, whichever comes first.

[0307] Randomization: Average tumor size approximately 135 mm 3 Randomization was performed once the target number of mice was reached. A total of 72 mice were enrolled in the BR1282 model study and randomly assigned to nine groups of eight mice each. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0308] Administration of test sample: The test sample was administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Treatment was initiated on the same day as randomization. The medication was administered in a laminar flow cabinet.

[0309] Observation and Data Collection: After inoculation with tumor cells, the morbidity and mortality rates of the animals are checked daily. At the point of routine monitoring, the animals are checked for adverse effects of tumor growth and treatment on normal behavior, including mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair matting, and any other abnormal effects. Tumor volume is measured two-dimensionally every 3-4 days using calipers, and the volume data is expressed in mm³ using the formula V=(L×W×W) / 2, 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). Medication, as well as tumor and weight measurements, are performed in a laminar flow cabinet. [Table 7]

[0310] The time-dependent tumor volume and mouse body weight in the efficacy study using the above test items are shown in Figures 13A and 13B.

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

[0312] 7-9 week old BALB / c nude female mice were given 5 × 10 in 0.1 ml of PBS mixed with Matrigel (1:1) to induce tumor formation. 6 Individual tumor cells were subcutaneously injected into the right anterior flank region. The tumor was 100-200 mm in size. 3 Once the mice reached their average size, they were randomized into five groups of eight mice each, and treatment was initiated. Randomization was performed using the "matched distribution" method (Study Director™ software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0313] Electronic caliper measurements were performed twice a week. Clinical signs, food and water consumption, and behavioral changes were observed daily, and the animals' body weight was measured twice a week. The evaluation item for the experiment was 3,000 mm 3 This is defined as the tumor volume, a weight loss of more than 20%, or weight loss over 28 days, whichever comes first.

[0314] Randomization: Average tumor size is approximately 100-200 mm 3 Randomization was performed once the target number of mice was reached. A total of 40 mice were enrolled in the NCI-H446 model study and randomly assigned to five groups of eight mice each. Randomization was performed using the "matched distribution" method (Study Director® software, version 3.1.399.19). The date of randomization is indicated as day 0.

[0315] Administration of test samples: ADC test samples were administered via intravenous injection through the tail vein at a dose of 10 mL / kg. Only single doses of ADC were initiated on the same day as randomization. Olaparib was administered orally at a dose of 50 mg / kg QD × 21 days, also initiated on the same day as randomization. Medication was administered in a laminar flow cabinet.

[0316] Observation and Data Collection: After inoculation with tumor cells, the morbidity and mortality rates of the animals will be checked daily. At the time of routine monitoring, the animals will be checked for tumor growth and adverse effects of the treatment on normal behavior, including mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair matting, and any other abnormal effects. Tumor volume will be measured in two dimensions every 3-4 days using calipers, and the volume data will be measured in mm using the formula V=(L×W×W) / 2. 3 It is expressed as follows, 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). Medication, as well as tumor and weight measurements, are performed in a laminar flow cabinet. [Table 8]

[0317] The time-dependent tumor volume and mouse body weight in the efficacy study using the above test items are shown in Figures 14A and 14B.

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

[0319] Randomization: In the tolerability trial, a total of 15 rats were enrolled and randomly assigned to five study groups of three rats each. Randomization was performed using the "matched distribution" method (StudyDirector® software, version 3.1.399.19). The date of randomization was indicated as day 0.

[0320] Administration of test samples: Treatment was initiated on the same day as randomization for each study design (Day 0). The selected dose levels were 75, 100, 125, and 150 mg / kg as a single dose. All animals were administered by slow intravenous injection (10 mL / kg).

[0321] Observation and Data Collection: Mortality and mortality rates of animals were checked daily. During routine monitoring, animals were checked for the effects of treatment on behaviors such as mobility, food and water consumption, weight gain / loss, eye / hair matting, and any other abnormalities. Mortality rates and observed clinical signs of individual animals were recorded in detail. Administration and weight measurements were performed in a laminar flow cabinet. Weight was measured daily using StudyDirector® software (version 3.1.399.19).

[0322] Figure 15 shows the time course of rat body weight for 12C6-3 as a test item at several dose levels.

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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