Homogeneous antibody-conjugates with high payload loading

Abstract

The present invention relates to homogeneous antibody-conjugates with high payload loading (high DAR) obtained by site-specific conjugation with a single antibody N-glycan. The conjugates according to the invention are homogeneous, i.e., with a DAR equal to or close to the theoretical DAR and narrow distribution, and do not require any genetic modification to the antibody. The invention also relates to a modular, non-genetic preparation method for such a conjugate, which method comprises three simple steps and can start from any antibody. These steps are (a) enzymatic remodeling of the glycan to give antibodies functionalised per antibody with two or four click probes, (b) strain-promoted cycloaddition with a multivalent bifunctional reagent comprising a cyclic alkyne and at least two click probes that are not reactive to the cyclic alkyne, and (c) strain-promoted cycloaddition with a multivalent bifunctional reagent comprising at least one cyclic alkyne and at least two click probes that are not reactive to the cyclic alkyne. And (c) subjecting the click probes to reverse electron demanding Diels-Alder cycloaddition with branched linker-drug constructs comprising a cyclic alkyne or strained olefin, linked to one or more payloads, preferably by cleavable linkers. The resulting conjugates have a DAR of 6 or higher, are rapidly generated, have high homogeneity and surprising stability. In addition, the HIC profile of the resulting ADC shows a relatively short retention time, and thus shows a very high potential in targeting tumor cells and / or treating cancer.

Description

Technical Field

[0001] This invention belongs to the medical field. More specifically, this invention relates to antibody-drug conjugates with high effective payload capacity, which are homogeneous and do not require genetic antibody modification. Such antibody-drug conjugates can be used to treat diseases more effectively, particularly cancer. Background Technology

[0002] Antibody-drug conjugates (ADCs) are considered a major class of targeted therapies, consisting of an antibody and a drug formulation attached to it. Antibodies (also called binders or ligands) can be in the form of small proteins (scFv, Fab fragments, DARPin, affinity molecules, etc.), but are typically selected IgG-type monoclonal antibodies (mAbs) based on their high selectivity and affinity for specific antigens, their long circulating half-life, and minimal immunogenicity. Therefore, mAbs, as ligands against carefully selected biological receptors, provide an ideal targeting platform for the selective delivery of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic agents to tumors via binding, internalization, intracellular processing, and ultimately the release of active catabolites. The cytotoxic agents can be small molecule toxins, protein toxins, or other forms (such as oligonucleotides). Thus, tumor cells can be selectively destroyed while normal cells not targeted by the antibody are preserved. Similarly, the chemical conjugation of antibacterial drugs (antibiotics) with antibodies can be used to treat bacterial infections, while conjugates of anti-inflammatory drugs are being investigated for the treatment of autoimmune diseases. For example, attaching oligonucleotides to antibodies is a promising approach for treating neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to selected specific cellular sites is a powerful approach to treating a variety of diseases, offering many advantages over systemic delivery of the same drug.

[0003] ADCs are prepared by conjugating a linker-drug to a protein, a process known as bioconjugation. Many techniques are known for bioconjugation, as summarized in G.H. Hermanson, “Bioconjugate Techniques,” Elsevier, 3rd edition, 2013 (incorporated by reference). Conceptually, the preparation of ADCs via bioconjugation requires the reaction of x reactive moieties F on an antibody with complementary reactive moieties Q on a drug (payload). See [link to previous section]. Figure 1 .

[0004] Typically, a chemical linker exists between the antibody (Q) and the payload. This linker needs to possess several key properties, including the requirement of long-term stability in plasma after drug administration. A stable linker enables the ADC to localize to the intended site or cell in vivo and prevents premature release of the payload in circulation, which indiscriminately induces various undesirable biological responses, thereby reducing the therapeutic index of the ADC. After internalization, the ADC should be processed to allow for efficient release of the payload, enabling it to exert its mechanism of action within the cell. The linker may also contain a spacer element. There are two types of linkers: non-cleavable linkers and cleavable linkers. Non-cleavable linkers consist of an atomic chain between the antibody and the payload, and are completely stable under physiological conditions regardless of the organ or biological compartment where the antibody-drug conjugate is located. Therefore, the release of the payload from an ADC with a non-cleavable linker depends on the complete (lysosomal) degradation of the antibody after the ADC is internalized into the cell. As a result of this degradation, the payload is released, still carrying the linker, as well as peptide fragments and / or amino acids from the antibody to which the linker was originally attached. Cleavable linkers utilize the inherent properties of cells or cellular compartments to selectively release the payload from ADCs, typically without leaving a trace of the linker after treatment. Three common mechanisms for cleavable linkers are: (1) susceptibility to specific enzymes, (2) pH sensitivity, and (3) sensitivity to the redox state of the cell (or its microenvironment). Cleavable linkers may also contain, for example, a self-immolative unit based on a p-aminobenzyl alcohol group or p-hydroxybenzyl alcohol and its derivatives and / or analogs, or a cyclized linker based on, for example, a 1,2-diaminoethane carbamate derivative. Linkers may also contain additional elements, often referred to as spacer units or extension units, to link the linker to a reactive group for attachment to the antibody via the reactive moiety F present on the antibody.

[0005] The reactive moiety F can be naturally present in the antibody. For example, the reactive moiety can be a side chain of lysine or cysteine, which can be used for acylation (lysine side chain) or alkylation (cysteine ​​side chain).

[0006] Acylation of the ε-amino group in the lysine side chain is typically achieved by subjecting the protein to treatment with reagents based on activated ester or activated carbonate derivatives, such as SMCC used in the manufacture of... Given that a given antibody may contain 60-90 lysine residues, the vast majority of which will exhibit reactivity to the acylation agent, precise titration of the acylation agent is required. However, such titration still results in highly heterogeneous conjugate mixtures, and the drug loading is merely an average of the random distribution of the drug attached to the antibody. For example, It has an average drug-antibody ratio (DAR) of approximately 4, but is actually composed of a mixture of components with DARs of 0-12. Adding a larger amount of acylation agent will result in a higher average DAR, for example, DAR 6 or DAR 8 can be achieved based on a random distribution containing even higher DAR species (i.e., >DAR 12).

[0007] Several reagents are known to be used for the alkylation of thiol groups in the side chain of cysteine; see [link to relevant documentation]. Figure 2 In cysteine ​​alkylation strategies, the vast majority are based on the use of maleimide reagents, for example, in the manufacture of... and The maleimide reagents used in this study. In addition to the standard maleimide reagents, a range of maleimide variants can also be used for more stable cysteine ​​coupling, as demonstrated by James Christie et al., J. Contr. Rel. [Journal of Controlled Release] 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol. [Nature Biotechnology] 2014, 32, 1059-1062 (both are incorporated herein by reference). Other methods of cysteine ​​alkylation include nucleophilic substitution, for example, with haloacetamides (typically bromoacetamide or iodoacetamide), see, for example, Alley et al., Bioconj. Chem. 2008, 19, 759-765, which is incorporated by reference, or various methods based on nucleophilic addition of unsaturated bonds, such as reactions with acrylate reagents, see, for example, Bernardim et al., Nat. Commun. 2016, 7, 13128 and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both of which are incorporated by reference, with phosphonamidat esters. For reactions e), see, for example, Kasper et al., Angew. Chem. Int. Ed. [Applied Chemistry International Edition] 2019, 58, 11625-11630, which is incorporated by reference; for reactions with allenamide, see, for example, Abbas et al., Angew. Chem. Int. Ed. [Applied Chemistry International Edition] 2014, 53, 7491-7494, which is incorporated by reference; for reactions with cyanoacetylene reagents, see, for example, Kolodych et al., Bioconj. Chem. [Bioconjugated Chemistry] 2015, 26, 197-200, which is incorporated by reference; for reactions with vinyl sulfone, see, for example, Gil de Montes et al., Chem. Sci. [Chemical Science] 2019, 10, 4515-4522, which is incorporated by reference, or for reactions with vinylpyridine, see, for example, Seki et al., Chem. Sci. [Chemical Science], 2021, 12, 9060-9068 and https: / / iksuda.com / science / permalink / (Accessed on July 26, 2020).

[0008] In the case of DAR, similar to lysine conjugation, this is controlled by titrating the alkylating agent used to react with the free cysteine ​​side chains (released by reducing interchain disulfides with, for example, TCEP or DTT). The final DAR is typically an average of a random mixture of different components, again similar to lysine conjugation. However, some significant differences can be noted: (a) different DAR types are typically composed of two (i.e., 2, 4, 6, 8), and (b) the maximum achievable DAR is 8 (if all released interchain cysteine ​​side chains have reacted). This also means that a homogeneous DAR8 ADC can be achieved through complete alkylation of all interchain cysteine ​​side chains. This is by far the most common method for generating DAR8 ADCs, and possibly the only method used for any clinical ADC with DAR8. It must be noted that such a method cannot generate a homogeneous DAR6 ADC or an ADC with DAR>8 unless specific cysteines are engineered or added to the antibody sequence using recombinant DNA technology. It must also be noted that any method that includes a reduction step can lead to antibody degradation (reduction of additional disulfide bonds) or fragment disorder (e.g., due to light chain exchange).

[0009] Alternative methods for antibody-chain disulfide bridge coupling include the use of cysteine ​​crosslinking agents, i.e., agents that react simultaneously with two cysteine ​​side chains. Examples of such crosslinking agents are disulfone agents, see, for example, Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both of which are incorporated herein by reference; mono- or dibromomaleimides, see, for example, Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269, both of which are incorporated herein by reference; bismaleimide agents, see, for example, WO 2014114207; bis(phenylthio)maleimide, see, for example, Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521, both of which are incorporated by reference; dibromopyridazine dione, see, for example, Robinson et al., RSC Advances 2017, 7, 9073-9077, which are incorporated by reference; bis(halomethyl)benzene, see, for example, Ramos-Tomillero et al., Bioconj. Chem. 2018, 29, 1199-1208, which are incorporated by reference; or other bis(halomethyl)aromatic compounds, see, for example, WO2013173391. Typically, the drug antibody loading of ADCs prepared by cysteine ​​crosslinking is 4 (DAR4), which is achieved by the complete alkylation of all cysteine ​​side chains released by reduction.

[0010] Another useful technique for coupling with cysteine ​​side chains is to form new disulfide bonds (i.e., asymmetric disulfide bonds, where one of the thiols is part of a good leaving group) by treating the released cysteine ​​side chain with a thiolizing agent, thereby forming a bioactive link that has been used to reversibly link protein toxins, chemotherapeutic agents, and probes to carrier molecules (see, for example, Pillow et al., Chem. Sci. 2017, 8, 366-370, which is incorporated herein by reference). Similar to cysteine ​​alkylation, the average DAR of such ADCs can be tuned to around 2–8 in the case of reduction of the native interchain disulfide bonds.

[0011] In addition to side-chain coupling with naturally occurring amino acids lysine or cysteine, a range of other coupling techniques have been explored based on a two-stage strategy, including (a) introducing a novel reactive group F followed by (b) reacting it with another complementary reactive group Q. For example, a method can be used to introduce a given number of reactive moieties F onto an antibody; this number could be two, four, or eight. See [link to relevant documentation]. Figure 3 .

[0012] Examples of non-natural reactive functional groups F that can be used for linker-drug bioconjugation are oxime groups (suitable for oxime linkages) or azide groups (suitable for click chemical conjugation). Oximes can be installed in antibodies by genetically encoding non-natural amino acids (e.g., acetylphenylalanine) (as demonstrated by Axup et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences] 2012, 109, 16101-16106 (incorporated by reference)) or by enzymatically alkylating cysteine ​​present in the CAAX sequence with an isoprenyl group containing a long-range ketone group (as disclosed, for example, in WO 2012153193). Azides can be genetically encoded in antibodies as either azidomethylphenylalanine or azidophenylalanine, as demonstrated, for example, by Axup et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences] 2012, 109, 16101-16106 (incorporated by reference). Similarly, Zimmerman et al., Bioconj. Chem. [Bioconjugated Chemistry] 2014, 25, 351-361 (incorporated by reference) introduced azidomethylphenylalanine (AzPhe) into monoclonal antibodies using a cell-free protein synthesis method for conversion into ADCs via metal-free click chemistry. Furthermore, Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097 (incorporated by reference), demonstrated that methionine analogs (such as azidehomalanine (Aha)) can be introduced into proteins via auxotrophic bacteria and further converted into protein conjugates via click chemistry. Finally, Nguyen et al., J. Am. Chem. Soc. 2009, 131, 8720-8721 (incorporated by reference), demonstrated the use of pyrrolidone-lysyl-tRNA synthetase / tRNA. CUAThe genetic encoding of aliphatic azides in recombinant proteins was performed, and tagging was achieved via click chemistry (through copper-catalyzed alkyne-azide cycloaddition (CuAAC) or strain-promoted alkyne-azide cycloaddition (SPAAC)). Furthermore, CuAAC and SPAAC, i.e., linker-drug bioconjugation with antibodies (and other biomolecules such as glycans and nucleic acids), can be achieved through a range of other metal-free click chemistry methods; see, for example, Nguyen and Prescher, Nature. Rev. Chem. [Nature Review Chemistry] 2020, 4, 476-489, which is incorporated by reference. For example, the oxidation of certain tyrosine residues in proteins can produce ortho-quinones, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes; see, for example, Bruins et al., Chem. Eur. J. [Chemical-European Journal] 2017, 24, 4749-4756, which is incorporated by reference. In addition to cyclooctynes, certain cycloheptynes ​​are also suitable for metal-free click chemistry, as reported by Wetering et al., Chem. Sci. [Chemical Science] 2020, 11, 9011-9016 (which is incorporated by reference). Tetraazine moieties can also be introduced into proteins or glycans through various means (e.g., genetic encoding or chemical acylation) and can also undergo cycloaddition with cyclic alkenes and alkynes. Figure 4 A list of functional group F and Q pairs for metal-free click chemistry is provided.

[0013] In SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne, and the cycloaddition with the azide-modified antibody is driven by the relief of the cyclostrain. Conversely, the linker-drug can be functionalized with azides, and the antibody can be functionalized with a cyclic alkyne. Figure 5 Various strained alkynes applicable to metal-free click chemistry are shown.

[0014] A growing trend in the field of ADCs is the enzymatic installation of non-natural functional groups F. For example, Lhospice et al., Mol. Pharmaceut. [Molecular Pharmaceutics] 2015, 12, 1863-1871 (incorporated by reference), utilized bacterial transglutaminase (BTG or TG enzyme) to install the azide moiety onto antibodies. Cheng et al., Mol. Cancer Therap. [Molecular Cancer Therapy] 2018, 17, 2665-2675 (incorporated by reference), reported a genetic approach based on C-terminal TG enzyme-mediated azide introduction, followed by conversion to ADCs via metal-free click chemistry.

[0015] In WO 2014065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12, and Wijdeven et al., MAbs 2022, 14, 2078466 (all incorporated by reference), demonstrated that enzymatic remodeling of native antibody glycans at N297 can introduce azido-modified sugars suitable for attaching cytotoxic payloads using metal-free click chemistry. See also Figure 6 Similarly, an enzymatic polysaccharide remodeling scheme can be used to attach free thiol groups to the antibody for coupling based on any of the cysteine ​​coupling methods described above.

[0016] β-glucuronidase is an enzyme that plays a role in the breakdown of endogenous glucuronic acid-containing glycosaminoglycans and is typically located in lysosomes of cells, including lysosomes in first-pass tissues such as the liver and intestines. It has been reported that β-glucuronidase concentrations are higher in many solid tumors (including lung, breast, and gastrointestinal cancers) and in the tumor microenvironment than in normal tissues, and this enzyme is not detected in systemic circulation. Furthermore, the levels of these enzymes are elevated in necrotic areas of tumors due to the release of lysosomal enzymes from dead cells into extracellular regions. Lysosomal β-glucuronidase concentrations are also high in inflammatory immune cells (including neutrophils and eosinophils), and these cells can release the enzyme at sites of inflammation, such as necrotic areas of human tumors. Overexpression of β-glucuronidase in certain tumors is a well-known approach in oncology research. Triggers cleavable by β-glucuronidase have been used for anthracyclines, auristatin, bacitracin, and camptothecin, as well as multi-compound prodrug release methods, PBD prodrugs, and ADCs. Similarly, β-galactosidase is an enzyme upregulated in the tumor microenvironment and can be used to selectively release payloads capped with β-galactosides attached to phenolic sites (e.g., p-hydroxybenzyloxycarbonyl groups).

[0017] Other enzymes upregulated in the tumor microenvironment (including cathepsins, matrix metalloproteinases (MMPs), legumains, and serine proteases such as elastases) have also been considered for releasing cytotoxic payloads, as summarized, for example, by M. Poreba, FEBS J. [FEBS Journal], 2020, 287, 1936-1969. Research on these two TME-associated enzymes has led to the development of cleavable linkers based on specific peptide sequences (e.g., Val-Cit or Val-Ala for cathepsins, Pro-Leu-Gly (PLG) for MMPs, Asn-Asn, Ala-Asn, or Asn-Ala for legumains, and Asn-Pro-Val (NPV) for serine proteases such as elastases).

[0018] Currently, cytotoxic payloads include, for example, microtubule disruptors [e.g., aurestatins such as monomethylaurestatin E (MMAE) and monomethylaurestatin F (MMAF), maytansine alkaloids such as DM1 and DM4, and tubulolysin], and DNA damaging agents [e.g., cazithromycin, pyrrolobenzodiazepines]. (PBD) dimer, indolinebenzodiazepine (indolinobenzodiapine) dimer, benzomycin, anthracyclines such as PNU-159,682, topoisomerase inhibitors [e.g., DXd, eczema, SN-38], or RNA polymerase II inhibitors [e.g., muscarine]. Approved ADCs include payloads such as MMAE, MMAF, DM1, DM4, chalcogenide, SN-38, DXd, and PBD dimer, and a benzomycin-based ADC is undergoing pivotal trials. More payloads (e.g., eribulin, indolinobenzodiazepine) are also available. Dimers, PNU-159,682, muscarine, and hammetrine are still under clinical evaluation or have undergone clinical trials in the past. Finally, various ADCs in early clinical or late preclinical stages are conjugated with novel payloads (e.g., KSP inhibitors, MMAD, cryptophytic acid, etc.). Various other payloads (e.g., doxorubicin, vinca alkaloids, and pyruvic acid SA) have also been evaluated in the past, but have been abandoned due to insufficient efficacy. For the same reason (lack of sufficient efficacy), even payloads with lower efficacy have not yet been clinically evaluated.

[0019] One way to expand the available payload range of an ADC by using drugs with lower potency than those in the current drug library is to increase the ADC's DAR, thereby effectively delivering more drug to the tumor in each internalization event. For example, Merca Therapeutics has developed a technology that supposedly enables an ADC's DAR of 12-15. This technology has been applied to its lead project, XMT-1536 (UpRi). To achieve such high DAR, a polymerized, highly polar linker technology was first developed to accommodate large amounts of hydrophobic payloads and prevent aggregation / degradation of the high-DAR ADC, which is essential to avoid severely impairing the pharmacokinetic behavior of the high-DAR ADC. Indeed, Hamblett et al., Clin. Canc. Res. [Clinical Cancer Research] 2004, 10, 7063-7070 (incorporated by reference), have convincingly demonstrated that DAR8 ADCs exhibit better in vitro efficacy due to their higher drug loading, while performing worse in vivo than equivalent DAR4 ADCs because of the significantly faster clearance of DAR8 species. Such poor PK characteristics can be clearly seen by measuring the chromatogram using hydrophilic interaction chromatography (HIC), especially the relatively long retention time compared to naked antibodies, which can be used as an indicator of poor PK performance, as disclosed by Burke et al., Mol. Canc. Ther. [Molecular Cancer Therapeutics] 2017, 16, 116-123 (incorporated by reference). Other solutions for mitigating undesirable PK characteristics and achieving DAR8 ADCs typically involve inserting branched hydrophilic spacers to shield hydrophobic payloads, such as those based on polyethylene glycol (PEG) (as disclosed by Burke et al., Mol. Canc. Ther. [Molecular Cancer Therapeutics] 2017, 16, 116-123 (incorporated by reference), used in SGN-CD228A and SGN-CD48A), polysarcosine (as disclosed by Viricel et al., Chem. Sci. [Chemical Science] 2019, 10, 4048-4053), or polysaccharides such as chitosan (as disclosed, for example, WO 2022058548 and WO 2022048883 and used in M1231).

[0020] To date, all high-DAR ADCs (approximately DAR8 or higher) that have entered clinical trials have been based on conjugation technology, which employs cysteine ​​alkylation with a linear linker-drug. Therefore, this technology is limited at DAR8 unless additional cysteines are engineered into the antibody via recombinant DNA technology. XMT-1536, with a drug loading of 12-15, is an exception to this rule; however, XMT-1536 is also a highly heterogeneous ADC, consisting of randomly conjugated cysteines and highly heterogeneous polymers attached to each cysteine. Summary of the Invention

[0021] The inventors have, for the first time, been able to prepare homogeneous antibody-conjugates with high payload capacity (high DAR) by site-specific conjugation with a single antibody N-glycan. The conjugates according to the invention are homogeneous, i.e., have a narrowly distributed DAR equal to or close to the theoretical DAR, and require no genetic modification of the antibody. The payload-to-antibody ratio (DAR) of the conjugates according to the invention is 6 or higher, preferably 8 or higher. Conjugates with a DAR up to 64 can be readily prepared.

[0022] The inventors have developed a modular, non-genetic preparation method for such conjugates, which comprises three simple steps and can begin with any antibody (see [link to method]). Figure 7 The inventors have developed a modular, non-genetic preparation method for such conjugates, comprising three simple steps and capable of starting with any antibody. These steps are (a) enzymatic remodeling of the glycan to give each antibody functionalized with two or four click probes (e.g., azides), and (b) a click reaction with a multivalent bifunctional reagent (e.g., strain-promoted azide-alkyne cycloaddition) comprising one click probe (e.g., a cyclic alkyne) reactive to the remodeled antibody and at least two click probes (e.g., tetrazine) unreactive to the other click probes (see Figure 8-). Figure 11 (c) a separate click reaction of the click probe with a branched linker-drug construct (e.g., inverse electron-demand Diels-Alder cycloaddition), which comprises a click probe (e.g., a cyclic alkyne or strained olefin) reactive to a previously non-reactive click probe, linked to one or more payloads, preferably via a cleavable linker (see [link to relevant documentation]). Figures 12-15The resulting conjugates have a DAR of 6 or higher, are rapidly generated, highly homogeneous, close to the theoretical DAR value, and exhibit surprising stability. Furthermore, the native glycosylation sites of the antibody are used for site-specific conjugation, thus eliminating the need for genetic engineering of the antibody to obtain conjugates with high DAR. Additionally, the resulting ADC's HIC profile, as well as in vitro and in vivo efficacy and tolerability studies, show relatively short retention times, thus demonstrating high potential in cancer therapy. Detailed Implementation definition

[0023] The verb “to comprise” and its variations, as used in this specification and claims, are used in their non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned.

[0024] Furthermore, mentioning an element with the indefinite article "a / an" does not preclude the possibility that there may be more than one / an element, unless the context explicitly requires the presence of one / an and only one / an element. Therefore, the indefinite article "a / an" usually means "at least one / an".

[0025] This document defines a linker as a portion that connects (covalently links) two or more elements of a compound. A linker may contain one or more spacer portions. This document defines a spacer portion as a portion that separates (i.e. provides distance between) two (or more) portions of the linker and covalently links them together. A linker may be, for example, part of a linker-conjugate, a linker-drug, a linker-payload (e.g., a linker-drug), or an antibody-conjugate, as defined below.

[0026] This document defines a “hydrophilic group” or “polar linker” as any molecular structure containing one or more polar functional groups that impart improved polarity to the attached molecule, and thus improve its water solubility. 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, acylsulfonamide groups, or carbamoylsulfonamide groups. In addition to higher solubility, other benefits of hydrophilic groups include improved click-coupling efficiency and, once bound to antibody-drug conjugates, reduced aggregation, improved pharmacokinetics, thereby enhancing efficacy and in vivo tolerability.

[0027] The term "its salt" refers to a compound formed when an acidic proton (usually the proton of an acid) is replaced by a cation (such as a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts not intended for administration to patients. For example, in a salt of a compound, the compound may be protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid is the anionic component of the salt. The term "pharmaceutically acceptable" salt means a salt that is acceptable for administration to patients (e.g., mammals) (a salt with an anti-charge ion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases, and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of compounds derived from various organic and inorganic anti-charge ions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium salts; and, when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochlorides, hydrobromic acid salts, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc.

[0028] The terms “enediyne” or “enediyne antibiotic” or “enediyne-containing cytotoxin” refer to any cytotoxin known in the art characterized by its presence as part of a cyclic molecule by the structural feature of 3-ene-1,5-diyne, and include neocytotoxicin (NCS), C-1027, cardaxetine (KED), maduratide (MDP), N1999A2, sporolide (SPO), cyanosporaside (CYA and CYN), as well as figilide, chachiin (CAL), esperamycin (ESP), danendomycin (DYN), namenamicin, shishijimicin, and uncialamycin (UCM).

[0029] As used herein, the term "alkylamino sugar" refers to a tetrahydropyranyl moiety that is linked to an alcohol functional group at its 2-position to form an acetal functional group, and is further substituted by at least one N-alkylamino group at the 3, 4, or 5-position. In this context, "N-alkylamino group" refers to an amino group having a methyl, ethyl, or 2-propyl group.

[0030] The term "click probe" refers to a functional part capable of undergoing a click reaction, i.e., two compatible click probes click each other, such that they are covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of this invention, a click probe Q in a compound according to the invention is capable of reacting with a click probe F on a (modified) protein, such that a coupling is formed upon a click reaction, wherein the protein is coupled to a compound according to the invention. Hereinafter, F and Q are compatible click probes. Click reactions are known in the art and generally refer to cycloaddition reactions, such as [4+2] cycloadditions (e.g., Diels-Alder, anti-electron Diels-Alder) and [3+2] cycloadditions (e.g., 1,3-dipolar cycloaddition). In the context of this invention, the term click reaction may also be referred to as cycloaddition.

[0031] The term "(hetero)alkyl" refers to both alkyl groups and heteroalkyl groups. A heteroalkyl group is one in which one or more carbon units (e.g., CH2, CH, or C) in an alkyl chain are surrounded by heteroatoms (e.g., O, S, S(O), S(O)2, or NR). 4 The alkyl group is replaced by another alkyl group. In other words, the alkyl chain is selected from O, S, S(O), S(O)2, and NR. 4 One or more elements are interrupted. Such interruptions differ from substituents because they occur within the chain of an alkyl group, while substituents are side-chain groups attached in a monovalent form to, for example, a carbon atom of an alkyl chain. In preferred embodiments, the (hetero)alkyl group is an alkyl group, such as ethyl (Et), isopropyl (i-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu), or n-pentyl.

[0032] Similarly, the term "(hetero)aryl" refers to both aryl groups and heteroaryl groups. A heteroaryl group is one in which one or more carbon units (e.g., CH) in a ring are surrounded by heteroatoms (such as O, S, N, or NR). 4 ) Replaced aryl groups.

[0033] In this document, "acylsulfonamide moiety" is defined as a sulfonamide moiety (H2NSO2NH2) that is N-acylated or N-carbamylated at one end of the molecule and N-alkylated (mono- or di-) at the other end of the molecule. In the context of this invention, and especially in the examples, this group is also referred to as "HS".

[0034] A "domain" can be any region of a protein, is typically defined based on sequence homology, and is usually associated with a specific structural or functional entity. Members of the CEACAM family are known to consist of Ig-like domains. In this document, the term "domain" is used to refer to a single Ig-like domain, such as an "N-domain," or a continuous group of domains, such as an "A3-B3 domain."

[0035] A "coding sequence" or sequence that "encodes" 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. In other words, the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. The coding sequence of a protein may include a start codon (usually ATG) and a stop codon.

[0036] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence, containing all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences such as promoter sequences that determine, for example, the conditions for gene expression. Some genes are not structural genes and may be transcribed from DNA into RNA but not translated into amino acid sequences. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term "gene" can refer to a genomic sequence that encodes a protein, i.e., a sequence containing regulators, promoters, introns, and exons.

[0037] The term "glycoprotein" is used in its normal scientific sense herein and refers to a protein containing one or more monosaccharide or oligosaccharide chains ("glycans") covalently bound to the protein. Glycans can be attached to hydroxyl groups on the protein (O-linked glycans), such as those attached to serine, threonine, tyrosine, hydroxylysine, or hydroxyproline; or to amide functional groups on the protein (N-glycoproteins), such as asparagine or arginine; or to carbon groups on the protein (C-glycoproteins), such as tryptophan. Glycans can contain more than one type of glycan, can contain combinations of one or more monosaccharides and one or more oligosaccharides, and can contain combinations of N-linked, O-linked, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and can therefore be considered glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antarcticis lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0038] The term "glycan" is used herein in its normal scientific sense and refers to a chain of monosaccharides or oligosaccharides linked to a protein. Therefore, the term "glycan" refers to the carbohydrate moiety of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of a sugar, which may be unsubstituted (monosaccharide) or may be further substituted at one or more hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain 1 to approximately 10 sugar moieties. However, when a longer sugar chain is linked to a protein, that sugar chain is also considered a glycan herein. The glycans of glycoproteins can be monosaccharides. Typically, the monosaccharide glycans of glycoproteins consist of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man), or fucose (Fuc) covalently attached to the protein. Glycans can also be oligosaccharides. The oligosaccharide chains of glycoproteins can be linear or branched. In oligosaccharides, the sugar directly attached to the protein is called the core sugar. In oligosaccharides, the sugar not directly attached to the protein but attached to at least two other sugars is called the internal sugar. In oligosaccharides, a sugar that is not directly attached to a protein but is attached to a single other sugar—that is, a sugar that does not carry additional sugar substituents at one or more of its other hydroxyl groups—is called a terminal sugar. To avoid confusion, a glycoprotein oligosaccharide may contain multiple terminal sugars, but only one core sugar. Glycans can be O-linked, N-linked, or C-linked. In O-linked glycans, monosaccharides or oligosaccharides typically bind to the O atom of a protein amino acid via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, monosaccharides or oligosaccharides bind to the protein via the N atom of a protein amino acid, typically via the amide nitrogen in the asparagine (Asn) or arginine (Arg) side chain. In C-linked glycans, monosaccharides or oligosaccharides bind to the C atom of a protein amino acid, typically the C atom of tryptophan (Trp).

[0039] The term “antibody” (AB) is used herein in its normal scientific sense. An antibody is a protein produced by the immune system that recognizes and binds to a specific antigen. An antibody is an example of a glycoprotein. The term “antibody” is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term “antibody” is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term “antibody” is intended to include the whole antibody, but also includes antibody fragments such as antibody Fab fragments, F(ab')2, Fv or Fc fragments derived from cleaved antibodies, scFv-Fc fragments, microantibodies, biantibodies, or scFv. Furthermore, the term also includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0040] Antibodies can be natural or conventional, in which two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: λ(l) and κ(k). A light chain comprises two domains or regions: a variable domain (VL) and a constant domain (CL). A heavy chain comprises four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions (VL and VH) of both the light and heavy chains determine the binding recognition and specificity with the antigen. The constant domains (CL and CH) of both the light and heavy chains 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 an immunoglobulin, consisting of a light chain and a variable portion of a heavy chain. Immunoglobulins can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), subclass, or allotype (e.g., human G1m1, G1m2, Gm3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of immunoglobulin molecules. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2, or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. Antibodies can be engineered to enhance or weaken binding to the Fc-γ receptor, as summarized by Saunders et al., Front. Immunol. [Immunology Frontiers] 2019, 10, doi:10.3389 / fimmu.2019.01296 and Ward et al., Mol. Immunol. [Molecular Immunology] 2015, 67, 131-141. For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. The binding to the Fc-γ receptor can also be eliminated by mutating the N297 amino acid to any amino acid other than asparagine, by mutating the T299 amino acid to any amino acid other than threonine or serine, or by enzymatically deglycosylating or trimming fully glycosylated antibodies using enzymes such as PNG enzyme F or glycosidases. Immunoglobulins can be derived from any species, including human, mouse, or rabbit.Each chain contains a different sequence structure field.

[0041] The percentage of "sequence identity" can be determined by comparing two sequences that align best on a comparison window, where the polynucleotide or polypeptide sequence portion of the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions) to allow for optimal alignment of the two sequences. A sequence that is "at least 85% identical to the reference sequence" is one that has 85% or more sequence identity over its entire length with the entire length of the reference sequence, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0042] The term "CDR" refers to the complementarity-determining region: antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed primarily of residues from the hypervariable region or complementarity-determining region (CDR). Occasionally, residues from the non-hypervariable region or framework region (FR) can affect the overall domain structure, thus influencing the binding site. Therefore, the complementarity-determining region, or CDR, refers to the amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, a typical antibody-antigen binding site comprises six CDRs, including CDR groups from the respective V regions of the heavy and light chains.

[0043] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule that targets a single amino acid sequence of a specific antigen and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced from a single B cell clone or hybridoma, but they can also be recombinant, i.e., produced through protein engineering.

[0044] The term "chimeric antibody" refers to an engineered antibody that, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In the examples, the chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal, and the CH and CL domains of another antibody (human antibody in the examples). Any animal, such as a mouse, rat, hamster, or rabbit, can be used as the non-human animal. Chimeric antibodies can also represent multispecific antibodies that are specific to at least two different antigens.

[0045] The term "humanized antibody" refers to an antibody that is wholly or partially non-human in origin and has been modified to replace certain amino acids, such as in the frame regions of the VH and VL domains, to avoid or minimize human immune responses. The constant domains of humanized antibodies are mostly human CH and CL domains. A "fragment" of a (conventional) antibody contains a portion of the complete antibody, specifically the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab′)2, Fab′, dsFv, (dsFv)2, scFv, sc(Fv)2, biantibodies, bispecific antibodies formed from antibody fragments, and multispecific antibodies. Fragments of conventional antibodies can also be single-domain antibodies, such as heavy chain antibodies or VHH.

[0046] The term "multivalent" refers to a linker molecule or linker portion of a larger molecule having multiple linking groups. For linker molecules, these linking groups are formed by reactive groups capable of covalently attaching to other molecules (e.g., payloads or antibodies). For linker portions of larger molecules (e.g., conjugates), the linking groups are covalently attached to other portions of the molecule (e.g., payloads or antibodies). A divalent linker has two linking groups, a trivalent linker has three linking groups, and so on. In the context of this invention, divalent is also referred to as 2-valent, and trivalent as 3-valent, etc.

[0047] The term "heterofunctional" refers to a linker molecule or linker portion of a larger molecule that has different linking groups with different reactivity or obtained through different reactivity. Typically, such linking groups in heterofunctional molecules do not reactivity with each other. This invention

[0048] The inventors have, for the first time, been able to prepare homogeneous antibody-conjugates with high payload capacity (high DAR) by site-specific conjugation with a single antibody N-glycan. The conjugates according to the invention are homogeneous, i.e., have a narrowly distributed DAR equal to or close to the theoretical DAR, and require no genetic modification of the antibody. The payload-to-antibody ratio (DAR) of the conjugates according to the invention is 6 or higher, preferably 8 or higher. Conjugates with a DAR up to 64 can be readily prepared.

[0049] The inventors have developed a modular, non-genetic preparation method for such conjugates, comprising three simple steps and capable of starting with any antibody. These steps are: (a) enzymatic remodeling of the glycan to give each antibody functionalized with two or four click probes (e.g., azides); (b) a click reaction with a multivalent bifunctional reagent (e.g., strain-promoted azide-alkyne cycloaddition) comprising one click probe (e.g., a cyclic alkyne) reactive to the remodeled antibody and at least two click probes (e.g., tetrazines) unreactive to the other click probes; and (c) separate click reactions of the click probes with branched linker-drug constructs (e.g., anti-electron-demand Diels-Alder cycloaddition) comprising one click probe (e.g., a cyclic alkyne or strained olefin) reactive to the previously unreactive click probe, linked to one or more payloads, preferably via cleavable linkers. The resulting conjugates exhibit a DAR of 6 or higher, are rapidly generated, highly homogeneous, close to the theoretical DAR value, and surprisingly stable. Furthermore, the native glycosylation sites of the antibodies are used for site-specific conjugation, thus eliminating the need for genetic engineering of the antibodies to obtain conjugates with high DAR. Additionally, the resulting ADCs show relatively short retention times in their HIC profiles and in vitro and in vivo efficacy and tolerability studies, demonstrating significant potential for cancer treatment.

[0050] In a first aspect, the present invention relates to a method for preparing antibody-to-antibody conjugates with a payload-to-antibody ratio (DAR) of 6 or higher, the method comprising: (a) Provides a structure Ab(F) 1 ) z The modified antibody, where Ab is an antibody, z is 2 or 4, and F 1 It is a click probe; (b) Mix the modified antibody with z equivalents of Q 1 L A (F 2 ) y The reaction, in which Q 1 It is for F 1 Reactive click probe, L A It is a heterofunctional (y+1) valence linker, where y is 2, 3, or 4, and F 1 It is for Q 1 Non-reactive click probes to obtain structures with Ab(Z) 1 L A (F 2 ) y ) z The antibody-linker construct, in which Z 1 Through F1 and Q 1 Linking groups obtained from the reaction; (c) Make the antibody-linker construct with a z×y equivalent of Q 2 (L B )D x The reaction, in which Q 2 It is for F 2 Reactive click probe, L B It is a (x+1) valence linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is the effective load molecule, to obtain the structure Ab(Z). 1 L A (Z 2 (L B )D x ) y ) z Coupled with Z 2 Through F 2 and Q 2 Linking groups obtained from the reaction.

[0051] In a second aspect, the present invention relates to couplings obtainable by the method according to the first aspect of the invention. Alternatively, couplings according to the second aspect can also be defined as having the structure Ab(Z). 1 L A (Z 2 (L B )D x ) y ) z ,in: -Ab is an antibody; -Z 1 and Z 2 It is a linker group that can be obtained through a reaction between two click probes; -x is 1, 2, 3 or 4; -y is 2, 3 or 4, where x+y is at least 4; -z is 2 or 4; -L A It is a heterofunctional (y+1) valence linker; and -L B It is a heterofunctional (x+1) valence linker; and -D represents the payload.

[0052] The conjugate according to the second aspect of the invention is ideally suited for targeting tumor cells, and particularly suitable for treating cancer. Therefore, the invention also relates to a method for targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting a conjugate according to the second aspect of the invention with cells that may express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor. Similarly, the invention also relates to a method for treating cancer, the method comprising administering a conjugate according to the second aspect of the invention to a subject in need, wherein cancer cells specifically express the extracellular receptor.

[0053] According to an aspect of the invention, the invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a conjugate according to a second aspect of the invention.

[0054] In a third aspect, the present invention relates to a structure Q 1 L A (F 2 ) y The heterofunctional connecton, wherein: -Q 1 It is a click probe; -L A It is a heterofunctional (y+1) valence linker; -y is 2, 3, or 4; and -F 1 It is for Q 1 Non-reactive click probes.

[0055] The heterobifunctional linker according to the third aspect of the invention is ideally suited as an intermediate for synthesizing the conjugate according to the second aspect.

[0056] The present invention also considers salts of conjugates and heterobifunctional linkers according to the invention, preferably pharmaceutically acceptable salts.

[0057] In the following text, conjugates according to the second aspect are first defined. The structural features of conjugates according to the invention also apply to heterobifunctional linkers according to the invention and methods for preparing antibody-loaded conjugates according to the invention. Those skilled in the art will understand that the structural features of conjugates according to the invention also apply to heterobifunctional linkers according to the invention. Those skilled in the art will understand that any structural features that remain unchanged in the coupling reaction are identically defined for each molecule according to the invention. In the coupling reaction, only the reactive portions F and Q are converted to the linking group Z. As those skilled in the art will understand, the definitions of the chemical portions and their preferred embodiments apply to all aspects of the invention. Coupler with a universal structure (2)

[0058] This invention relates to couplings having a universal structure (2): Ab[Z 1 L A (Z 2 (L B )D x ) y ] z (2) in: -Ab is an antibody; -Z 1 and Z 2 It is a linker group that can be obtained through a reaction between two click probes; -x is 1, 2, 3 or 4; -y is 2, 3 or 4, where x+y is at least 4; -z is 2 or 4; -L A It is a heterofunctional (y+1) valence linker; and -L B It is a heterofunctional (x+1) valence linker; and -D represents the payload. Antibody Ab

[0059] Ab is an antibody. Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, biantibodies, microantibodies, affinity molecules, affylin, affimilin, atrimer, fynomer, cysteine ​​knot, DARPin, adnectin / centryin, knottin, anticalin, FN3, Kunitz domain, OBody, bicyclic peptides, and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of IgA, IgD, IgE, IgG, and IgM antibodies. Even more preferably, Ab is an IgG antibody. The IgG antibody can be any IgG isotype. The antibody can be any IgG isotype, such as IgG1, IgG2, IgI3, or IgG4. Preferably, Ab is a full-length antibody, but Ab can also be an Fc fragment.

[0060] Antibody Abs typically exhibit specificity for extracellular receptors on tumor cells, preferably selected from the group consisting of: 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, short proteoglycans, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / cohesin-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, C D33, CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, Sealin-18.2, Sealin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, Fibronectin, FLT3, Folate receptor α, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylate cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, connexin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tendin.

[0061] Antibodies can also be specific for extracellular proteins caused by viral infections (such as human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV)). Antibodies can also be specific for tumor-associated carbohydrate antigens (TACAs) selected from the following group: Tn, STn, T antigen, LDN, Lewis antigen. c (Le c ), sialic acid-Lewis c (SLe c ), 6-sialic acid-Lewis c (6SLe c ), LN, α-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis antigen a (Le a ), sialic acid-Lewis a (SLe a ), 6-sialic acid-Lewis a (6SLe a Louis b (Le b ), sialic acid-Lewis b (SLe b ), 6-sialic acid-Lewis b (6SLe b Louis x (Le x ), sialic acid-Lewis x (SLe x ), 6-sialic acid-Lewis x (6SLe x Louis y (Le y ), sialic acid-Lewis y (SLe y ), 6-sialic acid-Lewis y (6SLe y Antibodies can be specific to both extracellular proteins and TACA.

[0062] In this art, the number of payloads D attached to a single antibody is referred to as DAR (Drug-Antibody Ratio). Although DAR values ​​may be as high as 64 in the context of this invention, it is preferred that DAR be an even number in the range of 6-32, more preferably 6, 8, 10, or 12, even more preferably 8 or 12, and most preferably DAR = 8. It should be understood that these are theoretical DAR values, and in practice, DAR may deviate slightly from these values ​​due to incomplete coupling. Therefore, conjugates may be obtained in the form of a mixture of conjugates with different DARs. In such cases, DAR usually refers to the average DAR of the mixture. This is well known in the field of bioconjugation. The conjugation technique used in this invention is site-specific conjugation via antibody glycans and using a click reaction, thereby providing conjugates with a narrow distribution whose DAR values ​​are close to the theoretical values ​​and have a small standard deviation. For example, when the theoretical DAR is 8, it is easy to obtain DAR values ​​higher than 7.4 or even higher than 7.6, indicating that most of the antibody in the reaction mixture has reacted completely and the DAR is 8. There are no antibody conjugates with a DAR higher than 8. Therefore, the average DAR of the obtained conjugate is close to the theoretical value, preferably the average DAR is up to 15% lower than the theoretical DAR, more preferably the average DAR is up to 10% lower than the theoretical DAR, or even up to 5% lower than the theoretical DAR.

[0063] In one embodiment of the conjugate according to the invention, only conserved glycosylation sites are used for conjugation as described below, i.e., z = 2, and the antibody is functionalized with a payload D appearing 2 × x × y times. Herein, x is 1, 2, 3, or 4, and y is 2, 3, or 4, where x + y is at least 4. Therefore, x + y is an integer in the range of 4-8. The exact numbers of x and y determine the DAR. In a preferred embodiment, y = 2 and x = 1, 2, 3, or 4, preferably x = 2, 3, or 4, more preferably x = 2. In another preferred embodiment, x = 2 and y = 2, 3, or 4, preferably y = 2 or 4, more preferably y = 2. In yet another preferred embodiment, x = y, preferably x = y = 2 or 4, more preferably x = y = 2.

[0064] In alternative embodiments of the conjugate according to the invention, both the conserved glycosylation site and the second glycosylation site are used for conjugation as described below, i.e., z = 4, and the antibody is functionalized with a payload D appearing 4 × x × y times. Herein, x is 1, 2, 3, or 4, and y is 2, 3, or 4, where x + y is at least 4. Therefore, x + y is an integer in the range of 4-8. The exact numbers of x and y determine the DAR. In a preferred embodiment, y = 2 and x = 1, 2, 3, or 4, preferably x = 1, 2, or 3, more preferably x = 2. In another preferred embodiment, x = 1 and y = 2, 3, or 4, preferably y = 2 or 4, more preferably y = 2. In yet another preferred embodiment, x = y, preferably x = y = 2 or 4, more preferably x = y = 2.

[0065] With these values ​​of z, x, and y, couplings with DAR in the range of DAR 6-64 are easily formed. The couplings according to the invention have an even number of DARs. In a preferred embodiment, DAR = 6, where z = 2, x = 1, and y = 3. In another preferred embodiment, DAR = 8, where z = 2, x = 2, and y = 2. In another preferred embodiment, DAR = 8, where z = 2, x = 1, and y = 4. In another preferred embodiment, DAR = 12, where z = 2, x = 2, and y = 3. In another preferred embodiment, DAR = 16, where z = 2, x = 2, and y = 4. In another preferred embodiment, DAR = 32, where z = 2, x = 4, and y = 4. In another preferred embodiment, DAR = 24, where z = 2, x = 3, and y = 4, or where z = 2, x = 4, and y = 3. In another preferred embodiment, DAR = 12, where z = 4, x = 1, and y = 3. In another preferred embodiment, DAR = 16, where z = 4, x = 2, and y = 2. In another preferred embodiment, DAR = 16, where z = 4, x = 1, and y = 4. The preferred coupling has a DAR in the range of 6-32, more preferably in the range of 6-16, and even more preferably in the range of 6-12. In yet another preferred embodiment, DAR is at least 8, preferably in the range of 8-32, more preferably in the range of 8-16, and even more preferably in the range of 8-12. Most preferably, DAR = 8.

[0066] The coupling compound according to the present invention contains two linking groups Z. 1 It is formed during the click reaction, wherein the antibody has the structure Ab(F) 1 ) z Furthermore, the heterofunctional (y+1) valence linker construct possesses the structural Q. 1 L A (F 2 )y In the first click response, F 1 and Q 1 The reaction proceeds by forming a linking group Z. 1 In the antibody and z×y part F 2 Covalent connections are formed between them.

[0067] In this paper, "(y+1) valence" refers to the number of linkage sites, which are either reactive groups F or Q (before the reaction) or linker groups Z (after the reaction). Therefore, in the case of y=2, the linker construct contains a click probe Q. 1 and two click probes F 2 And the valence of the connector is 2 + 1 = 3. Therefore, when y = 2, L A It is trivalent. Therefore, in the case of y=3, the connector subconstruct contains a click probe Q. 1 and three click probes F 2 And the valence of the connector is 3 + 1 = 4. Therefore, when y = 3, L A It is tetravalent. Therefore, in the case of y=4, the connector subconstruct contains a click probe Q. 1 and four click probes F 2 And the valence of the connector is 4 + 1 = 5. Therefore, when y = 4, L A It is pentavalent.

[0068] Part of the antibody can be the click probe F 1 Or linking group Z 1 Linker L, which connects to the peptide portion of the cell binder 6 Preferably, the linking group Z 1 The polysaccharide of Ab is linked to the cell-binding agent CB. Therefore, the conjugate according to the invention is preferably represented by the following: Ab[(L 6 )Z 1 L A (Z 2 (L B )D x ) y )] z , (3) in: -L 6 Yes - GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’- where G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fuc, w is 0 or 1, w' is 0 or 1, and L 7 It is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. Connector L 6

[0069] Preferably, a connector L is present. 6 This allows for the introduction of reactive groups F at specific sites on the antibody. 1 For example, via artificially introduced reactive groups F. 1 This is the case with coupling, for example, using transglutaminase, sorting enzymes, or through enzymatic modification of the polysaccharide (e.g., glycosyltransferases or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosamine transferase). For example, a modified sugar residue S(F) can be introduced at the polysaccharide. 1 )2, thereby extending the glycan with a single monosaccharide residue S, which introduces two reactive groups F onto the antibody glycan. 1 In the most preferred embodiment, coupling occurs via the antibody's glycans. The coupling site is preferably located on the antibody's heavy chain.

[0070] All recombinant antibodies generated in mammalian host systems contain a conserved N-glycosylation site at or near the asparagine residue at position 297 (Kabat number) of the heavy chain. This site is modified by a complex type of glycan. The use of this naturally occurring glycosylation site of the antibody is preferred; however, other glycosylation sites, including artificially introduced ones, can also be used for the linker L. 6 The connection. Therefore, in a preferred embodiment, L 6 The antibody is linked to an amino acid located at position 250-350 of the heavy chain, preferably at position 280-310, more preferably at position 295-300, and most preferably at position 297. Using this conserved glycosylation position of the antibody, the resulting conjugate forms a symmetrical dimer, wherein each half of the antibody contains one F... 2 And two click probes F 2 This will form arms with three or more payloads. Some antibodies may have a second glycosylation site per half, which, in the z=2 embodiment, is not used as a conjugation site. A person skilled in the art can perform enzymatic conversion in such a way that conjugation is performed using only the major glycosylation site. Alternatively, a person skilled in the art can perform enzymatic conversion in such a way that conjugation is also performed using the second glycosylation site, thereby doubling the DAR of the antibody-drug conjugate. In this embodiment, z=4 and each half of the antibody contains F twice. 2And all four click probes F 2 This will form an arm with three or more payloads.

[0071] L 6 It connects Ab to F. 1 or Z 1 The connector, and is determined by -GlcNAc(Fuc). w -(G) j -S-(L 7 ) w’ - indicates that G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fuc, w is 0 or 1, w' is 0 or 1, and L 7 It is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. Usually, L... 6 It is at least partially formed from antibody glycans.

[0072] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan or a part thereof. Therefore, the glycan-GlcNAc(Fuc) w -(G) j - Typically derived from the original antibody, where GlcNAc is the N-acetylglucosamine moiety and Fuc is the fucose moiety. Fuc is usually bound to GlcNAc via an α-1,6-glycosidic bond. Normally, the antibody may or may not be fucosylated (w=1) or unfucosylated (w=0). In the context of this invention, the presence of the fucosylated moiety is irrelevant; similar effects can be obtained using fucosylated (w=1) and unfucosylated (w=0) antibody conjugates. The GlcNAc residue can also be referred to as the core-GlcNAc residue and is a monosaccharide directly attached to the antibody peptide moiety.

[0073] S can be directly connected to the core - GlcNAc(Fuc). w The part where j=0 means that the remainder of the glycan is attached to the core-GlcNAc(Fuc) before S attachment. w The glycan is removed from the core. Such pruning of the glycan is well known in the art and can be achieved by the action of glycosidases. Alternatively, the core-GlcNAc(Fuc) can be removed. w There are one or more monosaccharide residues between part (G) and S, where j is an integer in the range of 1-10, preferably j = 1-5. In a preferred embodiment, (G) j It is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer in the range of 2-5. In another preferred embodiment, (G)j It is a monosaccharide fraction containing j monosaccharide residues G, where j is 1. (G) j It is typically linked to GlcNAc(Fuc) via a β-1,4 bond. w The GlcNAc portion. In a preferred embodiment, j is 0, 1, 3, 4, or 5; more preferably, j is 0 or 1; and most preferably, j is 0.

[0074] Although any monosaccharide present in the polysaccharide can be used as G, each G is preferably individually selected from the group consisting of: galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and N-acetylneuraminic acid. More preferred options for G are galactose, N-acetylglucosamine, and mannose. In the case of j=1, it is preferred that G = galactose and S = N-acetylneuraminic acid.

[0075] When j is 3-10, (G) j It can be linear or branched. Branched oligosaccharides (G) j Preferred examples are shown below (a), (b), (c), (d), (e), (f), (h) and (h).

[0076] In (G) j When present and j>2, it is preferred that its terminal is GlcNAc or Gal, preferably GlcNAc. In other words, the monosaccharide residue directly linked to S is preferably GlcNAc or Gal. The presence of the GlcNAc moiety is beneficial for the synthesis of functionalized antibodies because the monosaccharide derivative S can be readily introduced to the terminal GlcNAc residue via glycosyltransferase. The presence of the Gal moiety is beneficial for the synthesis of functionalized antibodies because the monosaccharide derivative S sialic acid can be readily introduced to the terminal Gal residue via sialyltransferase. In (G) having structures (a)-(h) j In the preferred embodiments described above, the portion S can be linked to any terminal GlcNAc residue (i.e., not a residue with a wavy bond) that is linked to the core GlcNAc residue on the antibody.

[0077] Antibodies and antibody-drug conjugates with j = 0 or 1 show no binding or significantly reduced binding to the Fc-γ receptor, while antibodies and antibody-drug conjugates with j in the range of 4-10 do bind to the Fc-γ receptor. Therefore, by selecting a certain value of j, the desired degree of binding to the Fc-γ receptor can be obtained. Thus, it is preferred that j = 0, 1, 4, 5, 6, 7, 8, 9, or 10, more preferably j = 0, 1, 4, or 5, and most preferably the antibody is pruned and j = 0.

[0078] S is a sugar or a sugar derivative. The term "sugar derivative" is used herein to refer to a derivative of a monosaccharide (i.e., a monosaccharide containing substituents and / or functional groups). Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose, 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 Gal, GalNAc, and NeuNAc. In a particularly preferred embodiment, S is GalNAc.

[0079] Linking group Z 1 or reactive group F 1 It can be directly attached to S, or between S and Z. 1 or F 1 Connectors L can exist between them. 7 Therefore, L 7 It can exist (w' = 1 or 2) or not exist (w' = 0). Typically, each Z part can be connected via connector L. 7 Connected to S, therefore in one embodiment, w' = 0 for x. Preferably, L 7 There is no such connection and each connection part Z is directly attached to S. If there is, L 7 It can be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. In a preferred embodiment, x = 1 and w' = 0 or 1, and most preferably x = 1 and w' = 0. Linking group Z 1 and Z 2

[0080] Z 1 and Z 2 It is a linker group that covalently links the antibody to the payload of the conjugate according to the invention. The term "linker group" as used herein refers to a structural element generated by the reaction between Q and F, used to link one part of the conjugate to another part of the same conjugate. 1 Through Q 1 and F 1 The click reaction between them is formed. Similarly, Z 2 Through Q 2 and F 2 The interaction is formed by clicks between the two points. In this article, Z refers to Z0. 1 and Z 2 Q refers to Q1 and Q 2 And F refers to F 1 and F 2 .

[0081] Those skilled in the art will understand that the exact properties of the linking group depend on the exact structures of the click probes Q and F. Those skilled in the art are familiar with complementary click probes that are reactive with each other and form suitable reaction partners Q. 1 / F 1 and Q 2 / F 2 For example, when F contains an alkynyl group, the complementary group Q includes an azide group. For example, when F contains an alkynyl group, the complementary group Q includes an alkynyl group. For example, when F contains a cyclopropenyl group, a trans-cyclooctene group, a cycloheptyne group, or a cyclooctene group, the complementary group Q includes a tetrazinyl group. In these special cases, Z is merely an intermediate structure and will expend N2, thereby generating a dihydropyridazine (reacting with alkenes) or a pyridazine (reacting with alkynes), such as... Figure 4 As shown.

[0082] The linking group Z is obtained via a cycloaddition reaction, preferably wherein the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition. Coupling reactions via cycloaddition are known to those skilled in the art, and those skilled in the art can select appropriate reaction partners F and Q and will understand the properties of the resulting linking group Z. Preferred cycloadditions are [4+2]-cycloadditions (e.g., Diels-Alder reactions) or [3+2]-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the cycloaddition is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are electron-demanding Diels-Alder cycloadditions. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition. Cycloadditions (e.g., Diels-Alder reactions and 1,3-dipolar cycloadditions) are known in the art, and those skilled in the art know how to perform them.

[0083] Preferably, Z contains a moiety selected from the group consisting of: triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazoline, pyrazoline, piperazine, or pyridazine, more preferably triazole, isoxazoline, or pyridazine. The triazole moiety is particularly preferred to be present in Z. In one embodiment, Z comprises a (hetero)cyclic alkene moiety, i.e., formed from Q which comprises a (hetero)cyclic alkyne moiety. In alternative embodiments, Z comprises a (hetero)cyclic alkane moiety, i.e., formed from Q which comprises a (hetero)cyclic alkene moiety. In this document, an aromatic ring (such as a triazole ring) is considered a heterocyclic alkane ring because it is formed by the reaction of an alkyne moiety and an azide moiety.

[0084] In a preferred embodiment, Z has a structure (Z1): In this article, it is described as The key is either a single key or a double key. Additionally: - Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za)-(Zm) as defined below, wherein the carbon atom marked with ** corresponds to the depiction of ring Z fused in (Z1) as follows: The two carbon atoms in the bond; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -Y 2 It is C(R) 31 )2、O、S、S (+) R 31 S(O)R 31 S(O) = NR 31 or NR 31 S (+) Is with B (-) Mutually balanced cation sulfur atoms, of which B (-) It is an anion, and each R in it is an anion. 31 R alone 15 Or a connection to D (via L); -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u+u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; -v = integers in the range of 8-16; - The ring Z is formed by cycloaddition and is preferably selected from (Za)-(Zm).

[0085] In a preferred embodiment, u+u' = 0, 4, 5, 6, 7, or 8, more preferably 0, 4, or 5. If described as If the bond is a double bond, then u+u' = 4, 5, 6, 7, or 8 is preferred, and u+u' = 4 or 5 is more preferred. If described as... If the key is a single key, then u+u' = 0 or 5 is preferred. Preferably, the tilde marked with * is connected to CB, optionally via L. 6 The tilde marked with ** is connected to L.

[0086] Particularly preferred is Z comprising a (hetero)cyclic olefin moiety, i.e., described as The key It is a double bond. In a preferred embodiment, Z is selected from the structures (Z2)-(Z20) described below:

[0087] In this article, the connection with L is depicted using a tilde. B (-) It is an anion, preferably a pharmaceutically acceptable anion. B (+) It is a cation, preferably a pharmaceutically acceptable cation. Ring Z is formed by a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazoline, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za)-(Zm) as described below, wherein the carbon atom marked with ** corresponds to two carbon atoms of the (hetero)cycloalkane ring fused with ring Z in (Z2)-(Z20). Since, in the context of this embodiment, the linking group Z is formed by reacting with a (hetero)cycloalkyne, it is described above as The key is a double bond.

[0088] In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction, especially in the case of the formation of ring (Zl), is optimal. Therefore, in a preferred embodiment, ring Z is (Zl), wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0089] When Z contains a (hetero)cyclic olefin moiety, it is preferred that the ring Z is selected from (Za), (Zj), (Zk) or (Zl), and more preferably the ring Z is based on the structure (Za) or (Zl).

[0090] In another preferred embodiment, Z is selected from the structures (Z21)-(Z38a) described below:

[0091] In this document, the connection with L is depicted using a tilde. Structure (Z29) can be an internal or external configuration, preferably an internal configuration. In structure (Z38), B (-) It is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from the structure (Za)-(Zm) as defined above.

[0092] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structures (Z8), (Z26), (Z27), (Z28), (Z37), or (Z38a), which may optionally be substituted. Each of these preferred options for Z is further defined below.

[0093] Therefore, in a preferred embodiment, Z comprises the heterocyclic heptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocyclic heptene moiety according to structure (Z37) is not substituted.

[0094] In a preferred embodiment, Z comprises a heterocyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is not substituted. In the context of this embodiment, Z preferably comprises a heterocyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2). lAnd 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, even more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z is further defined below according to structure (Z42).

[0095] In alternative preferred embodiments, Z comprises a heterocyclooctene moiety according to structure (Z26), (Z27), or (Z28), which is optionally substituted. In the context of this embodiment, Z preferably comprises a heterocyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y 1 Is it O or NR? 11 , where R 11 Independently selected from the group consisting of: hydrogen, straight-chain or branched C1-C 12 alkyl groups or C4-C 12 (Hetero)aryl groups. The aromatic ring in (Z40) is optionally O-sulfated at one or more positions, while the ring in (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclic octene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), as further defined below.

[0096] In an alternative preferred embodiment, Z comprises a heterocyclic heptenyl group and is according to structure (Z37).

[0097] In a particularly preferred embodiment, Z comprises a cyclooctenyl group and is according to structure (Z42): In this article: - The key marked with * connects to CB, while the tilde key marked with ** connects to L; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C5-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -R 18 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -R 19 Choose from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, which are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N, and S, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are independently and optionally substituted, or R 19 It is the second occurrence of Z (or Q) or D connected via a gap; and -l is an integer in the range of 0 to 10.

[0098] In a preferred embodiment of the group according to structure (Z42), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1-C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably all R 15 All are H. In a preferred embodiment of the group according to structure (Z42), R 18 Independently selected from the group consisting of: hydrogen, C1-C6 alkyl groups, most preferably two R groups. 18 All are H. In a preferred embodiment of the group according to structure (Z42), R 19 It is H. In a preferred embodiment of the group according to structure (Z42), l is 0 or 1, more preferably l is 1.

[0099] In a particularly preferred embodiment, Z comprises a (hetero)cyclooctenyl group and is according to structure (Z43): In this article: - The key marked with * connects to CB, while the tilde key marked with ** connects to L; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C5-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -Y is N or CR 15 ; - The carbon atom in the fused aromatic ring can be replaced by a nitrogen atom as shown in (Z6a)-(Z6d), preferably where Y is CR. 15 .

[0100] In a preferred embodiment of the group according to structure (Z43), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -S(O)3 (-) C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1-C6 alkyl, more preferably R 15 Independently select from the following groups: hydrogen and -S(O)3 (-) In a preferred embodiment of the group according to structure (Z43), Y is N or CH, more preferably Y = N.

[0101] In a particularly preferred embodiment, Z comprises a heterocyclic heptenyl group and is according to structure (Z37) or (Z38a), wherein ring Z is a triazole.

[0102] In an alternative preferred embodiment, the linking group Z comprises a (hetero)cycloalkane moiety, i.e., described as The bond is a single bond. The (hetero)cycloalkane group can also be called a heterocycloalkyl group or cycloalkyl group, preferably a cycloalkyl group, wherein the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornenyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, all of which may be optionally substituted. Particularly preferred are the (hetero)cyclopropyl group, the (hetero)cycloheptyl group, or the (hetero)cyclooctyl group, wherein the (hetero)cyclopropyl group, the (hetero)cycloheptyl group, or the (hetero)cyclooctyl group is optionally substituted. Preferably, Z comprises a cyclopropyl moiety according to structure (Z44), a heterocyclic butane moiety according to structure (Z45), a norbornene or norbornene group according to structure (Z46), a (hetero)cyclic heptyl moiety according to structure (Z47), or a (hetero)cyclic octyl moiety according to structure (Z48). In this document, Y... 3 Selected from C(R) 23 2. NR 23 Or O, where each R 23 It is hydrogen alone, C1-C6 alkyl, or linked to L (optionally via a spacer), and is labeled with The bond is a single or double bond. In another preferred embodiment, the cyclopropyl group is according to structure (Z49). In yet another preferred embodiment, the (hetero)cycloheptyl group is according to structure (Z50) or (Z51). In yet another preferred embodiment, the (hetero)cyclooctyl group is according to structure (Z52), (Z53), (Z54), (Z55), or (Z56).

[0103] In this document, the R group on Si in (Z50) and (Z51) is typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is formed during a cycloaddition reaction and is generally selected from the structure (Zn)-(Zu), where the carbon atom marked with ** corresponds to the two carbon atoms of the (hetero)cycloalkane ring fused with ring Z in (Z44)-(Z56), while the carbon marked with * is connected to CB. Since, in the context of this embodiment, the linking group Z is formed by reaction with a (hetero)cycloalkene, it is described above as... The key is a single key.

[0104] In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction, especially in the case of the formation of the (Zu) ring, is optimal. Therefore, in a preferred embodiment, ring Z is (Zu), wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0105] When Z contains a (hetero)cycloalkane moiety, it is preferred that ring Z is selected from (Zn), (Zs), (Zt) or (Zu), and most preferably ring Z is according to the structure (Zu).

[0106] In a preferred embodiment, the linking group Z comprises a portion selected from (Z1)-(Z56), wherein the ring Z is selected from (Za)-(Zu).

[0107] In this invention, Z 1 and Z 2 The exact structure usually varies because Z 1 Through Q 1 and F 1 The reaction formed by Z, and 2 Through Q 2 and F 2 The reaction is formed. In this paper, F 2 Q 2 It is reactive, but to Q 1 It is not reactive, therefore Q 1 and Q 2 They should be different.

[0108] In a preferred embodiment, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is either tetrazine or nitroacetate, and Q 2It is a bicyclic nonyne or a cyclic olefin, such as trans-cyclooctene or cyclopropene. More preferably, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is tetrazine, and Q 2 It is a dicyclic nonyne.

[0109] In this article, F 1 and Q 1 The reaction will preferably form a linking group Z according to the structure (Z5), (Z6), (Z7), (Z11), (Z17), (Z18), (Z19), or (Z19a). 1 Wherein ring Z is based on structure (Za), preferably on structure (Z26), (Z27), (Z28), (Z32), (Z37), (Z38) or (Z38a), more preferably on structure (Z40), (Z41) or (Z43) or on structure (Z37) or (Z43). In this document, Q 2 Preferably, in this paper, F 2 and Q 2 The reaction will preferably form a linking group Z. 2 The linking group is based on structures (Z8), (Z44), (Z47), (Z48), (Z49), (Z54), (Z55), or (Z56), more preferably on structures (Z29), (Z48), or (Z49), and most preferably on structure (Z42). In this document, ring Z is based on structures (Zd), (Zl), (Zq), or (Zu), preferably on structure (Zl) or (Zu), and most preferably on structure (Zl). Alternatively, the linking group Z... 2 It is based on structure (Z8), wherein ring Z is based on structure (Z1), preferably on structure (Z29), and more preferably on structure (Z42). Connector L A

[0110] Connector L A via connector L B and linking group Z 2 The payload D and Ab are connected via the linker Z. 1 (In the couplings according to the invention) link, or attach the reactive group F 2 With reactive group Q 1 (In the connector according to the invention) connection. The connector is known in the art and can be cuttable or non-cuttable. Connector L A Preferably, it is not a cuttable connector, but connector L B Preferably, it is a cuttable connector.

[0111] Connector L A With Z appearing once 1 (or Q) 1 Z appears y times and y times. 2 (or F) 2 ) connection. Therefore, the connector L A The valence of is y+1 or "(y+1) valence". In this paper, "(y+1) valence" refers to the number of linkages, which are either reactive groups F or Q (before the reaction) or linking groups Z (after the reaction). Therefore, in the case of y=2, the linker is linked to a Z 1 / Q 1 and two Z 2 / F 2 The linker is connected, and the valence of the linker is 2 + 1 = 3. Therefore, when y = 2, L A It is trivalent. Therefore, in the case of y=3, the connector is connected to a Z. 1 / Q 1 and three Z 2 / F 2 The linker is connected, and the valence of the linker is 3 + 1 = 4. Therefore, when y = 3, L A It is tetravalent. Therefore, in the case of y=4, the connector is connected to a Z. 1 / Q 1 and four Z 2 / F 2 The linker is connected, and the valence of the linker is 4 + 1 = 5. Therefore, when y = 4, L A It is pentavalent.

[0112] Connector L A It can be called "heterofunctional," meaning it contains two different functions, referring to its relationship with Z. 1 / Q 1 and Z 2 / F 2 Different chemical connections.

[0113] In a preferred embodiment, L A It is a heterodifunctional linker with the following structure: -(L 11 )-BM(L 12 -) y in: -L 11 With Q 1 or Z 1 Connect, and each L 12 With F 2 or Z 2 Connection, where L 11 and L 12Each is individually composed of one or more structural units selected from the following: C(R) 13 )2、C(R 13 )=C(R 13 C≡C, C(O), NR 13 O, S, S(O), S(O)2, PR 13 and P(O)R 13 Preferably, these structural units are selected from CH2, CH=CH, C(O), and NR. 13 ,O,S,S(O) and S(O)2, - Each R 13 As defined below for structure (23), preferably R 13 It is H; and -BM stands for branch. Branches are further defined below.

[0114] In a more preferred embodiment, L A It is a structurally distinct bifunctional trivalent linker: -(L 11 )-BM(L 12 -)(L 13 -) in: -L 11 With Q 1 or Z 1 Connect, and L 12 and L 13 With F 2 or Z 2 Connection, where L 11 L 12 and L 13 Each is individually composed of one or more structural units selected from the following: C(R) 13 )2、C(R 13 )=C(R 13 C≡C, C(O), NR 13 O, S, S(O), S(O)2, PR 13 and P(O)R 13 Preferably, these structural units are selected from CH2, CH=CH, C(O), and NR. 13 , O, S, S(O) and S(O)2, and - Each R 13 As defined below for structure (23), preferably R 13 It is H; and -BM stands for branch. Branches are further defined below.

[0115] L 11 L12 and L 13 For example, you can choose from groups consisting of straight chains or branches C1-C. 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkylene groups, C8-C 200 cycloalkynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imidene group, C9-C 200 Arylynylene group. Optionally, the alkylene group, alkenylene group, ynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylynylene group can be substituted, and optionally, the group can be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably selected from the group consisting of: O, S(O). y’ and NR 21 Where y' is 0, 1, or 2, preferably y' = 2, and R 21 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups. In one embodiment, optional substituents may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamine, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, urethane groups, cyclodextrins, crown ethers, sugars (e.g., monosaccharides, oligosaccharides), phosphoric acid or its esters, phosphonic acid or esters, hypophosphonic acid or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or hyposulfonic acids.

[0116] In the context of this invention, having structure Q 1 L A (F 2 ) y Connector subconstructs and structure Ab(F) 1 ) z The reaction, thus the F of each antibody Ab 1 and Q 1 The reaction proceeds z times, thus forming z linker groups Z for each antibody Ab. 1After the reaction is complete, each antibody contains z×y click probes F. 2 The antibody-linker construct formed therefrom has the structure Ab(Z) 1 L A (F 2 ) y ) z . Branch section BM

[0117] In the context of this invention, a "branch portion" refers to a portion embedded in a connector that links the three portions. In other words, a branch portion contains at least three keys that connect to the other portions.

[0118] In the context of this invention, any portion containing at least three bonds connected to other portions is suitable as a branch portion. Suitable branch portions include carbon atoms (BM-1), nitrogen atoms (BM-3), phosphorus atoms (phosphine (BM-5) and phosphine oxide (BM-6)), aromatic rings such as phenyl rings (e.g., BM-7) or pyridyl rings (e.g., BM-9), (hetero)rings (e.g., BM-11 and BM-12), and polycyclic moieties (e.g., BM-13, BM-14, and BM-15). Preferably, BM is selected from carbon atoms, nitrogen atoms, phosphorus atoms, (hetero)aromatic rings, (hetero)rings, or polycyclic moieties, more preferably BM is a carbon atom or a nitrogen atom. When BM is a nitrogen atom, the branch nitrogen can be part of an amide group, for example via a C=O group and Z. 1 Or Q 1 The connection and the two substituents on nitrogen and the two occurrences of Z 2 or F 2 The connection. When BM is a carbon atom, the carbon atom is preferably part of a trivalent amino acid (e.g., lysine, aspartic acid, or glutamic acid).

[0119] The appropriate branch section BM is selected from the structures (BM-1) to (BM-15) described below, where three branches (i.e., the keys with the other sections as defined above) are indicated by * (keys marked with *).

[0120] In (BM-1), one of the branches marked with * can be a single bond or a double bond, using... Indication. In (BM-11) to (BM-15), the following applies: -n, p, q and q are each independently an integer in the range of 0-5, preferably 0 or 1, and most preferably 1; -W 1 W2 and W 3 Each of them is independently selected from C(R) 21 ) w and N; -W 4 W 5 and W 6 Each of them is independently selected from C(R) 21 ) w+1 、N(R 22 ) w O and S; -each Indicates a single bond or a double bond; -w is 0, 1, or 2, preferably 0 or 1; - Each R 21 Choose independently from the following groups: hydrogen, OH, Cl-C 24 Alkyl groups, C1-C 24 alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, wherein these C1-C 24 Alkyl groups, C1-C 24 alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S and NR. 3 One or more heteroatoms are interrupted, where R 3 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; and - Each R 22 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, wherein these C1-C 24 Alkyl groups, C1-C 24 alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S and NR. 3 One or more heteroatoms are interrupted, where R 3 It is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0121] Technicians understand the value of w and its origin. The bond order is interdependent. Therefore, whenever a W occurs and binds to an inner loop double bond, w = 1 for that occurrence of W, and whenever a W occurs and binds to two inner loop single bonds, w = 0 for that occurrence of W. For BM-12, at least one of o and p represents 0.

[0122] Representative examples of the branched portions of structures (BM-11) and (BM-12) include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, aziridine, aziridine, diazacyclobutane, diazacyclobutane, oxacyclobutane, thiohexacyclobutane, pyrrolidine, dihydropyrrolyl, tetrahydrofuranyl, dihydrofuranyl, thiohexacyclopentyl, imidazolinyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolinyl, isothiazolyl, dioxazolinyl, dioxazopentyl, dithiohexacyclopentyl, piperidinyl, oxacyclohexane, thiohexane, piperazine, morpholino, thiomorpholino, dioxacyclohexane, trioxacyclohexane, dithiazolinyl, trithiohexane, aziridine, oxacyclohexacyclolinyl, and thiohexacyclohexacyclolinyl. Preferred cyclic moieties used as branching sections include cyclopropenyl, cyclohexyl, oxacyclohexane (tetrahydropyran), and dioxacyclohexane. The substitution pattern of the three branches determines whether the branching section has a structure (BM-11) or a structure (BM-12).

[0123] Representative examples of the branches of the structure (BM-13) to (BM-15) include decahydronaphthalene, tetrahydronaphthalene, dihydronaphthalene, naphthalene, indene, dihydroindene, isoindene, indole, isoindole, indoline, isoindoline, etc.

[0124] In a preferred embodiment, BM is a carbon atom. When the carbon atom is according to structure (BM-1) and has all four bonds connected to different parts, the carbon atom is chiral. The stereochemistry of the carbon atom is not important to the invention and can be S or R. This also applies to phosphine (BM-6). Most preferably, the carbon atom is according to structure (BM-1). One of the branches indicated by * in the carbon atom according to structure (BM-1) can be a double bond, in which case the carbon atom can be part of an alkene or imine. When BM is a carbon atom, the carbon atom can be part of a larger functional group, such as acetal, ketal, hemiketal, oxoester, oxocarbonate, amino acid, etc. In this regard, preferred amino acids are Asp, Gly, Lys, and iGlu. This also applies when BM is a nitrogen or phosphorus atom, in which case it can be part of an amide, imide, imine, phosphine oxide (as in BM-6), or phosphate triester.

[0125] In a preferred embodiment, BM is a phenyl ring. Most preferably, the phenyl ring is according to structure (BM-7). The substitution mode of the phenyl ring can be of any regiochemistry type, such as a 1,2,3-substituted phenyl ring, a 1,2,4-substituted phenyl ring, or a 1,3,5-substituted phenyl ring. For optimal flexibility and conformational freedom, it is preferred that the phenyl ring is according to structure (BM-7), and most preferably the phenyl ring is 1,3,5-substituted. This also applies to pyridine rings having (BM-9).

[0126] In the preferred structure of the BM defined above, the BM typically contains three connection points. Those skilled in the art can determine corresponding BMs with four or five connection points. For example, (BM-1) and (BM-7)-(BM-15) are also suitable as BMs with more connection points. Alternatively, the connector may contain more than one BM to produce four or five connection points.

[0127] In a preferred embodiment, the branch portion BM is selected from carbon atoms, nitrogen atoms, phosphorus atoms, (hetero)aromatic rings, (hetero)cyclic or polycyclic portions, more preferably carbon atoms or nitrogen atoms. Connector L B

[0128] Connector L B via linker group Z 2 and connector L A Connect the payload D to Ab (in the coupling according to the invention), or connect the payload D to the reactive group Q. 2 (In the payload-connector construct) Connection. Connectors are known in the art and may be cuttable or non-cuttable. Connector L BPreferably, it is a cuttable connector, and the connector L A Preferably, it is not a cuttable connector.

[0129] Connector L B With Z appearing once 2 (or Q) 2 The connection L appears x times and is a D connection. Therefore, the connector L B The valence of Q is x+1 or "(x+1) valence". In this paper, "(x+1) valence" refers to the number of linkage points, which are the effective loads or reactive groups Q. 2 (Before reaction) or linking group Z 2 (After the reaction). Therefore, in the case of x=1, the connector is connected to a Z. 2 / Q 2 It is connected to a payload D, and the valence of the connector is 1 + 1 = 2. Therefore, when x = 1, L B It is divalent. Therefore, in the case of x = 2, the connector is connected to a Z. 2 / Q 2 It is connected to two payloads D, and the valence of the connector is 2 + 1 = 3. Therefore, when x = 2, L B It is trivalent. Therefore, in the case of x=3, the connector is connected to a Z. 2 / Q 2 It is connected to three payloads D, and the valence of the connector is 3 + 1 = 4. Therefore, when x = 3, L B It is tetravalent. Therefore, in the case of x=4, the connector is connected to a Z. 2 / Q 2 It is connected to four payloads D, and the valence of the connector is 4 + 1 = 5. Therefore, when x = 4, L B It is pentavalent.

[0130] Connector L B It can be called "heterofunctional," meaning it contains two different functions, referring to its relationship with Z. 2 / Q 2 A chemically different connection from D.

[0131] In a preferred embodiment, L B It is a heterodifunctional linker with the following structure: -(L 1 )-(BM) r’ -[(L 2 ) o -(L 3 ) p -(L 4 ) q -] x in: -L 1 With Q 2 or Z 2 Connect, and all occurrences of L 4 All are connected to D; -L 1 L 2 L 3 and L 4 Each individually is Q 2 or Z 2 Connector that is connected to D; -o, p, and q are each individually 0 or 1, preferably where o = p = 1; -r' is 0 or 1, where r' is 0 when x is 1, or r'1 is 1 when x is 2, 3 or 4; -BM stands for branch. Branches are further defined above.

[0132] In an alternative preferred embodiment, x = 1 and L B It is a heterofunctional bivalent linker with the following structure: -(L 1 )-(L 2 ) o -(L 3 ) p -(L 4 ) q - in: -L 1 With Q 2 or Z 2 Connect, and L 4 Connect to D; -L 1 L 2 L 3 and L 4 Each individually is Q 2 or Z 2 Connector that is connected to D; -o, p, and q are each individually 0 or 1, preferably where o = p = 1; -BM stands for branch. Branches are further defined above.

[0133] In a more preferred embodiment, x = 2 and L B It is a heterobifunctional trivalent linker with the following structure: -(L 1 )-(BM)'-[(L 2 ) o -(L 3 ) p-(L 4 ) q -]2 in: -L 1 With Q 2 or Z 2 Connect, and L 4 Connect to D; -L 1 L 2 L 3 and L 4 Each individually is Q 2 or Z 2 Connector that is connected to D; -o, p, and q are each individually 0 or 1, preferably where o = p = 1; -BM stands for branch. Branches are further defined above.

[0134] Alternatively, L B It is a heterodifunctional linker with the following structure: -(L 1 )-[(L 2 ) o -(L 3 ) p -(L 4 ) q -] x In the case of x = 2, 3, or 4, BM embeds the connector L. 1 .

[0135] In the context of this invention, having structure Q 2 (L B )D x Effective load - connecting sub-constructs with structure Ab(Z) 1 L A (F 2 ) y ) z The antibody-linker construct reaction, thereby enabling the F of each antibody Ab to... 2 and Q 2 The reaction proceeds z×y times, thus forming z×y linker groups Z for each antibody Ab. 2 After the reaction is complete, antibody-conjugates are formed, each containing z×y×x payloads D. The resulting antibody-conjugates have the structure Ab(Z) 1 L A (Z 2 (L B )D x ) y )z .

[0136] L 1 L 2 L 3 and L 4 These are connectors or connecting units, and each of o, p, and q is individually 0 or 1, preferably where o = p = 1. In a preferred embodiment, at least connector L is present. 1 and L 2 (That is, o = 1; p = 0 or 1; q = 0 or 1), more preferably, there exists a connector L. 1 L 2 and L 3 (That is, o = 1; p = 1; q = 0 or 1). In one embodiment, L 1 L 2 L 3 and L 4 Each is individually composed of one or more structural units selected from the following: (hetero)aryl, CH2, CH=CH, C≡C, C(O), NR 13 O, S, S(O), S(O)2, PR 13 and P(O)R 13 Preferably, these structural units are selected from aryl, CH2, CH=CH, C(O), and NR. 13 O, S, S(O) and S(O)2. The following provides L... 1 L 2 L 3 and L 4 Preferred embodiments of each of them.

[0137] Connectors, especially connector L 1 It may contain one or more branch points for attaching multiple payloads to a single linker group. In a preferred embodiment, the linker of the coupling according to the invention contains a branch portion. In the context of the invention, a "branch portion" refers to a portion embedded in the linker that connects the three portions. In other words, the branch portion contains at least three bonds connected to the other portions, typically one bond connected to Z. 1 Alternatively, a Q connection can be used, with one key connecting to payload D and another key connecting to a second payload D. If present, the branch portion is preferably embedded with a connector L. 1 In, more preferably Sp 3 Part of or as NR 13 The nitrogen atom. In the context of this invention, any portion containing at least three bonds connected to other portions is suitable as a branch portion. In a preferred embodiment, the branch portion is selected from carbon atoms, nitrogen atoms, phosphorus atoms, (hetero)aromatic rings, (hetero)cyclic or polycyclic portions. Most preferably, the branch portion is a nitrogen atom. Connector L 1

[0138] L 1 For example, you can choose from the following groups: straight chains or branches C1-C 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkylene groups, C8-C 200 cycloalkynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imidene group, C9-C 200 Arylynylene group. Optionally, the alkylene group, alkenylene group, ynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylynylene group can be substituted, and optionally, the group can be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably selected from the group consisting of: O, S(O). y’ and NR 21 Where y' is 0, 1, or 2, preferably y' = 2, and R 21 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups. In one embodiment, optional substituents may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamine, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, urethane groups, cyclodextrins, crown ethers, sugars (e.g., monosaccharides, oligosaccharides), phosphoric acid or its esters, phosphonic acid or esters, hypophosphonic acid or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or hyposulfonic acids.

[0139] In a preferred embodiment, connector L 1 It contains a polar group, which can also be present in L 1 In the chain. Such polar groups can be selected from (poly)ethylene glycol diamine (e.g., 1,8-diamino-3,6-dioxane or equivalents containing a longer ethylene glycol chain), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,x'-diaminoalkanes (where x' is the number of carbon atoms in the alkane, preferably x' = 1-10), -(O).a -C(O)-NH-S(O)2-NR 13 -(as further defined below, see structure (23)), -C(S(O)3 (-) )-、-C(C(O)2 (-) -, -S(O)2-, -P(O)2 (-) -、-O(CH2CH2O) t -、-NR 30 (CH2CH2NR 30 ) t - and the following two structures:

[0140] For the polar group defined above, which end is attached to Z? 1 Which end is connected to (L) 2 ) o It's irrelevant.

[0141] The polar group may also contain amino acids, preferably selected from Arg, Glu, Asp, Ser, and Thr. In this paper, R... 13 The structure (23) is further defined below. t is an integer in the range of 0-15, preferably 1-10, more preferably 2-5, and most preferably t = 2 or 4. Each R 30 H and C alone 1-12 Alkyl, C 1-12 Aryl, C 1-12 alkylaryl or C 1-12 Aryl group. Linker L 1 It may contain more than one such polar group, for example, at least two polar groups. Polar groups may also be present in the linker L. 1 In the branch, this branch originates from a branch portion defined elsewhere. In L 1 In this context, nitrogen or carbon atoms are preferably used as branching parts. Particularly preferred is the presence of -O (CH2CH2O) within the branch. t - Polar groups.

[0142] In a preferred embodiment, connector L 1 It is or contains a sulfonamide group, preferably a sulfonamide group according to structure (23):

[0143] The wavy line indicates a connection to the remainder of the compound, usually associated with Q. 2 or Z 2 and with L 2 L 3 L 4 Or a connection of D. Preferably, (O)a C(O) part and Q 2 or Z 2 Connection, and NR 13 Part of L 2 L 3 L 4 Or D connection, preferably with L 2 connect.

[0144] In structure (23), a = 0 or 1, preferably a = 1, and R 13 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, these C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Alternatively, R 13 It is optional via the interval portion, preferably via Sp as defined below. 2 In one embodiment, D, which is connected to N, is connected via -(B). e -(A) f -(B) g -C(O)- is connected to N. Alternatively, R 13 Optionally connected to other parts of the connector via spacers to form a ring structure. For example, R 13 It can be connected to the linker via the CH2CH2 spacer to form a piperazine ring, wherein the connection with D is via the second nitrogen of the piperazine ring.

[0145] In a preferred embodiment, R 13 It is hydrogen, C1-C 20 Alkyl groups, preferably C1-C 16 Alkyl groups, more preferably C1-C 10 An alkyl group, or optionally linked via a spacer portion to another D or to other locations on the linker. In this document, the alkyl group is optionally substituted and optionally selected from O, S, and NR. 14One or more heteroatoms of O are preferably interrupted, wherein R 14 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In another preferred embodiment, R 13 It is C1-C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, or even more preferably C1-C 10 An alkyl group, wherein the alkyl group is optionally interrupted by one or more O atoms, and wherein the alkyl group is optionally substituted with a -OH group, preferably a terminal -OH group. In this embodiment, R is further preferred. 13 It is a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 The group selected is from the group consisting of: hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl, or optionally linked via a spacer portion to another D or to other locations of the linker; more preferably selected is from the group consisting of: hydrogen, methyl, ethyl, n-propyl, and isopropyl, or optionally linked via a spacer portion to another D or to other locations of the linker; and even more preferably selected is from the group consisting of: hydrogen, methyl, and ethyl, or optionally linked via a spacer portion to another D or to other locations of the linker. Even more preferably, R... 13 It is hydrogen or optionally connected via a spacer portion to another D or to other locations connected to the linker, and most preferably R 13 It is hydrogen.

[0146] In a preferred embodiment, L 1 It is based on structure (24):

[0147] In this paper, a and R 13 As defined above, Sp 1 and Sp 2 Each is an independent spacer portion, and b and c are independently 0 or 1. Preferably, b = 0 or 1 and c = 1, more preferably b = 0 and c = 1. In one embodiment, the spacer Sp 1 and Sp 2 Independently select from the following groups: straight chain or branched chain C1-C 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkylene groups, C8-C 200 cycloalkynyl group, C7-C 200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C 200 aryl imide groups and C9-C 200 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, including alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups, may optionally be substituted. When alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkylene groups, cycloalkylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalene groups, and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, it is preferred that said groups are interrupted by one or more O atoms and / or by one or more SS groups.

[0148] More preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 100 alkylene groups, C2-C 100 imide groups, C2-C 100 Imynyl group, C3-C 100 Cycloalkyl groups, C5-C 100 Cycloalkylene groups, C8-C 100 cycloalkynyl group, C7-C 100 alkylarylene groups, C7-C 100 arylalkylene groups, C8-C 100 aryl imide groups and C9-C 100 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24alkynyl group and C3-C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0149] Even more preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 50 alkylene groups, C2-C 50 imide groups, C2-C 50 Imynyl group, C3-C 50 Cycloalkyl groups, C5-C 50 Cycloalkylene groups, C8-C 50 cycloalkynyl group, C7-C 50 alkylarylene groups, C7-C 50 arylalkylene groups, C8-C 50 aryl imide groups and C9-C 50 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0150] Even more preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 20 alkylene groups, C2-C 20 imide groups, C2-C 20 Imynyl group, C3-C 20 Cycloalkyl groups, C5-C 20 Cycloalkylene groups, C8-C 20 cycloalkynyl group, C7-C 20 alkylarylene groups, C7-C 20 arylalkylene groups, C8-C 20 aryl imide groups and C9-C 20The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0151] In these preferred embodiments, it is further preferred that the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalene group, and arylalkynylene group are unsubstituted and optionally selected from O, S, and NR. 16 One or more heteroatoms of the group, preferably O interrupted, wherein R 16 It is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0152] Most preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 20 Alkyl groups, which are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, including alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups, are optionally substituted. In this embodiment, it is further preferred that the alkylene groups are unsubstituted and optionally selected from O, S, and NR. 16 One or more heteroatoms of the group, preferably O and / or SS interrupted, wherein R 16 It is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0153] Therefore, the preferred interval portion Sp 1 and Sp 2 Includes -(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 - where r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, and even more preferably 1, 2, 3 or 4.

[0154] Alternatively, the preferred connector L 1 It can be made by -(W) k -(A) d -(B) e -(A) f -(C(O)) g - indicates that: -d = 0 or 1, preferably d = 1; -e = an integer in the range of 0-10, preferably e = 0, 1, 2, 3, 4, 5 or 6, more preferably an integer in the range of 1-10, and most preferably e = 1, 2, 3 or 4; -f = 0 or 1, preferably f = 0; -where d+e+f is at least 1, preferably in the range of 1-5; and preferably where 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 sulfonamide group based on structure (23); -B is a part of -CH2-CH2-O- or -O-CH2-CH2-, or (B) e It is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 The -CH2- part, where the definition of e1 is the same as that of e; -W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH2) m C(O)-、-C(O)(CH2) m C(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably where W is -OC(O)NH- or -C(O)(CH2). m C(O)NH- or -C(O)NH-, where m is an integer in the range of 0-10, preferably m = 0, 1, 2, 3, 4, 5 or 6, and most preferably m = 2 or 3; - Preferably, where L 1 Via (W) k Connected to Q and via (C(O)) g Preferably connected to L via C(O) 2 L 3 Or D, preferably connected to L 2 .

[0155] In the context of this embodiment, the wavy line in structure (23) indicates a junction with an adjacent group (e.g., (W)). k (B) e and (C(O)) g The connection of ) is preferred. A is based on structure (23), where a = 1 and R 13 =H or C1-C 20 Alkyl groups, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0156] Preferred connector L 1 It has a structure -(W) k -(A) d -(B) e -(A) f -(C(O)) g -,in: (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 or4, 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.

[0157] In this paper, it is preferred that when d and / or f = 1, then a = 1 and R 13 =H. The optimal choice is a connector structure (a).

[0158] In a preferred embodiment, connector L 1 It contains a branched nitrogen atom located at Q or Z and (L 2 ) o The main chain contains an additional portion of D as a substituent, which is preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR in structure (23). 13 , where R 13 It connects to the second D via the spacer portion. Alternatively, the branched nitrogen atom may be located according to the structure -(W). k -(A) d -(B) e -(A) f -(C(O))g -ofL 1 Inside. In one embodiment, L 1 By -(W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A) d -(B) e -(A) f -(C(O)) g -]2 indicates that A, B, W, d, e, f, g, and k are as defined above and are selected individually each time they appear, and BM is the branch portion, preferably a branched nitrogen atom, with two -(A) atoms. d -(B) e -(A) f -(C(O)) g - Connected to it. In this article, the two (C(O)) g All of them are related to -(L) 2 ) o -(L 3 ) p -(L 4 ) q -D connects, where L 2 L 3 L 4 , o, p, q, and D are as defined above and each is selected individually. In a preferred embodiment, L 2 L 3 L 4 Each of , o, p, q and D for (C(O)) g Both parts of the connection are identical.

[0159] Preferred linker L containing branched nitrogen atoms 1 It has a structure -(W) k -(A) d -(B) e -(A) f -(C(O)) g -N[-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, where: (i) k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is based on structure (23), where a = 0 and R 13 =H; e = 1, 2, 3 or 4, preferably e = 2. (j)k=d=g=e'=g'=1;f=d'=0;W=-C(O)-;B=B'=-CH2-CH2-O-;A is based on structure (23), where a=0 and R 13 =H; e = 1, 2, 3 or 4, preferably e = 2. Connector L 2

[0160] Connector L 2 It is a peptide spacer. Linker L 2 It either does not exist (o = 0) or it exists (o = 1). Preferably, the connector L 2 It exists and o=1. Peptide spacer L 2 and cuttable connector L 3 The combination of these is well known in the art. Connector L 2 More preferably, peptides are recognized and cleaved by specific cleavage enzymes, serving as both recognition and cleavage sites. This allows cleavage to occur in specific environments (e.g., specific tumors) where these cleavage enzymes are expressed. Because different peptide sequences are cleaved by different enzymes, L... 2 The group also allows for the customization of conjugates for specific therapies. The peptide sequence can be cleaved by intracellular and / or extracellular enzymes.

[0161] Peptide spacers can also be defined as (NH-CR) 17 -CO) n , where R 17 This refers to an amino acid side chain as known in the art. This definition also covers proline, which has an R atom linked to a nitrogen atom to form a cyclic moiety. 17 In this document, amino acids can be natural or synthetic. Preferably, all amino acids are in the L configuration. n is an integer in the range of 1-5, preferably 2-4. Therefore, the peptide spacer contains 1-5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3), or tetrapeptide (n=4), and most preferably, the peptide spacer is a dipeptide. R 17 The term "amino acid side chain" is preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolidone, 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 Val, Cit, Ala, Glu, and Lys. In other words, R 17Preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp), and CH2(1H-indole-3-yl)(Trp). 17 Particularly preferred embodiments are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). Most preferably, R 17 It is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0162] Although any peptide spacer can be used, it is preferred that the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Glu-Gly-Cit, Glu-Gly-Val, Asp-Val-Cit, and iGlu-Val-Cit. ,Phe-Cit,Phe-Ala,Phe-Lys,Phe-Arg,Ala-Lys,Leu-Cit,Ile-Cit,Trp-Cit,Asn-Asn,Ala-Ala-Asn,Ala-A sn, Asn-Ala, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly, Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, Phe-Gly, Phe-Gly, Ser-Gly, Gly-Phe-Gly, Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly, Phe-Gly-Gly-Gly, Gly-Gl y-Gly-Phe, Phe-Phe-Gly-Gly, Gly-Gly-Phe-Phe, Gly-Gly-Gly-Phe-Gly and Lys, more preferably selected from Val-Cit, Val-Ala, Glu -Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably selected from Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala, Asn-Asn, Ala-Ala-Asn, Asn-Ala, most preferably selected from Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala, or Asn-Ala. In this document, AcLys is eN-acetyllysine and iGlu is isoglutamate. In one embodiment, L 2 =Val-Cit. In another embodiment, L 2 =Val-Ala. In another embodiment, L 2 =Asn-Ala. In another embodiment, L 2 =Glu-Gly-Cit. In another embodiment, L 2 =Glu-Val-Ala.

[0163] In one embodiment, amino acid side chain R 17The substance is replaced by a polar group, preferably selected from oxo groups, (poly)ethylene glycol diamine, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, sugars (e.g., monosaccharides, oligosaccharides), phosphoric acid or its esters, phosphonic acid or esters, hypophosphonic acid or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or hyposulfonic acids.

[0164] In a particularly preferred embodiment, L 2 Contains peptide spacers represented by a universal structure (25), preferably L 2 Represented by the general structure (25):

[0165] In this article, R 17 As defined above, preferably R 17 It is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates that it is related to (L) 1 ) n and (L) 3 ) p The connection, preferably, is based on the L of structure (25). 2 Connected to (L) via NH 1 ) n And connected to (L) via C(O) 3 ) p . Connector L 3

[0166] Connector L 3 It is a self-cutting spacer, also known as a self-destructing spacer. Connector L 3 It either does not exist (p=0) or it exists (p=1). Preferably, the connector L 3 There exists and p = 1. L 2 The cutting caused the connector L 3 The elimination of 1,6-β in L leads to decarboxylation and release of the payload D. This is beneficial for increasing the ability to cleave L 2 The probability of releasing the payload in the region of enzyme overexpression. Furthermore, payload release can induce bystander killing, which is advantageous because not all cancer cells in a tumor overexpress the target receptor. Therefore, in this embodiment, the linker L is preferred. 2 and L 3 Both exist (o = p = 1).

[0167] Preferably, L 3It is a p-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (26):

[0168] In this article, the wavy line represents L. 1 or L 2 and L 4 Or the connection of D. Typically, PABC derivatives are connected via NH and L. 1 or L 2 Connection, preferably with L 2 Connect, and via OC(O) to L 4 Or D connection.

[0169] Ring A is a 5- or 6-membered aromatic ring or a heteroaromatic ring, preferably a 6-membered aromatic ring or a heteroaromatic ring. Suitable 5-membered rings are oxazoles, thiazoles, and furans. Suitable 6-membered rings are phenyl and pyridyl groups. Ring A can be selected from halogens, X... 2 R 4 、N(R 4 2. C 1-4 Alkyl groups and NO2 substituents are used for substitution. In this paper, X... 2 and R 4 As defined above, including preferred embodiments thereof. In preferred embodiments, optional substituents are selected from F, Cl, Br, OH, OR. 4 SH, NH2, Et, Me, and NO2. In a particularly preferred embodiment, ring A contains 0-2 substituents, more preferably 0 or 1 substituent, and most preferably ring A is unsubstituted. In a preferred embodiment, ring A is 1,4-phenyl, 1,2-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0170] R 21 Selected from H, R 26 C(O)OH and C(O)R 26 , where R 26 It is C1-C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl group, C3-C 10 alkyl(hetero)aryl groups and C3-C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally selected from O, S, and NR. 28 One or more heteroatoms are interrupted, where R 28 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, R 26 It is C3-C 10(Hetero)cycloalkyl or polyalkylene glycols. The polyalkylene glycols are preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O). s H or -(CH2CH2CH2O) s H. The most preferred polyalkylene glycol is polyethylene glycol, preferably -(CH2CH2O). s H, where s is an integer in the range of 1-10, preferably 1-5, and most preferably s = 1, 2, 3, or 4. More preferably, R 21 Is it H or C(O)R 26 , where R 26 =4-methylpiperazine or morpholine. Most preferably, R 21 It is H.

[0171] In an alternative embodiment, connector L 3 There exists (p=1) and connector L 2 It does not exist (o = 0), and L 3 It is a glucuronide-m-amide-benzyloxycarbonyl derivative, preferably a glucuronide derivative according to structure (27):

[0172] In this article, the wavy line represents L. 1 and L 4 Or the connection of D. Typically, glucuronide derivatives are linked via NH and L. 1 Connect, and via (O)CO and L 4 Or D connection. Rings A and R 21 This is as defined for PABC derivatives according to structure (26). Preferably, ring A is a 6-membered aromatic or heteroaromatic ring, such as an oxazole, thiazole, furan, phenyl, or pyridyl. In preferred embodiments, ring A is 1,3,4-phenyl, 2,4,5-pyridyl, or 2,5,6-pyridyl. Most preferably, A is 1,3,4-phenyl. Preferably, R... 21 Is it H or C(O)R 26 , where R 26 =4-methylpiperazine or morpholine. Most preferably, R 21 It is H.

[0173] According to the connector L of structure (27) 3It can be cleaved by β-glucuronidase, similar to the mechanism in PABC, which leads to the self-destruction, decarboxylation, and release of the payload of the p-hydroxybenzyloxy group. ADCs containing glucuronide derivatives according to structure (27) are particularly useful for treating cancers with β-glucuronidase overexpression. It has been reported that the concentration of β-glucuronidase in many solid tumors (including lung cancer, breast cancer, and gastrointestinal cancer) and in the tumor microenvironment is higher than that in normal tissues, and the enzyme is not detected in systemic circulation. Therefore, conjugates according to the invention containing glucuronide derivatives according to structure (27) are preferred for treating patients with lung cancer, breast cancer, and gastrointestinal cancer. Connector L 4

[0174] Connector L 4 It either does not exist (q=0) or it exists (q=1). Preferably, the connector L 4 It exists and q = 1. Connector L 4 Selected from: -Based on structure-NR 22 -(C x -alkylene)-C(O)-aminoalkanoic acid spacers, where x is an integer in the range of 1-20, and R 22 It is an H or C1-C4 alkyl group; -Based on structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 -C(O)- ethylene glycol spacer, where e6 is an integer in the range of 1-10, e7 is an integer in the range of 1-3, and R 22 It is H or C1-C4 alkyl; and -Based on structure-NR 22 -(C x -alkylene)-NR 22 -(C(O)) h - a diamine spacer, where h is 0 or 1, x is an integer in the range of 1-20, and R 22 It is H or C1-C4 alkyl.

[0175] Connector L 4 It can be an aminoalkanoic acid spacer, i.e., -NR 22 -(C x -alkylene)-C(O)-, where x is an integer in the range of 1 to 20, preferably 1 to 10, and most preferably 1 to 6. In this document, the aminoalkanoic acid spacer is typically connected via a nitrogen atom and an L... 3 It is connected to D via a carbonyl moiety. Preferred linker L. 4The aminocaproic acid is selected from 6-aminocaproic acid (Ahx, x=5), β-alanine (x=2), and glycine (Gly, x=1), and even more preferably 6-aminocaproic acid or glycine. In one embodiment, L 4 =6-Aminohexanoic acid. In one embodiment, L 4 = Glycine. In this article, R 22 It is H or C1-C4 alkyl, preferably R. 22 It is H or methyl, most preferably R. 22 It is H.

[0176] Alternatively, connector L 4 It can be based on structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 -(C(O)- ethylene glycol spacer, wherein e6 is an integer in the range of 1-10, preferably in the range of 2-6, and e7 is an integer in the range of 1-3, preferably e7 is 2. In this document, R 22 It is H or C1-C4 alkyl, preferably R. 22 It is H or methyl, most preferably R. 22 It is H.

[0177] Alternatively, connector L 4 It can be based on structure-NR 22 -(C x -alkylene)-NR 22 -(C(O)) h - a diamine spacer, wherein h is 0 or 1, x is an integer in the range of 1-20, preferably an integer in the range of 2-6, even more preferably x = 2 or 5, and most preferably x = 2. R 22 It is H or C1-C4 alkyl. In this article, R 22 It is H or C1-C4 alkyl, preferably R. 22 It is H or methyl, most preferably R. 22 It is a methyl group. In this text, h is preferably 1, in which case the linker L... 4 It is particularly suitable for coupling via phenolic hydroxyl groups present on payload D. Payload D

[0178] D, also known in the art as "payload," refers to the compound linked to or to be linked to the antibody Ab. Payload molecules are well known in the art, particularly in the field of antibody-drug conjugates, as portions covalently attached to the antibody and released from the antibody upon uptake of the conjugate and / or cleavage of the linker. In preferred embodiments, the payload is selected from the group consisting of: active substances, reporter molecules, polymers, solid surfaces, hydrogels, nanoparticles, microparticles, and biomolecules. Particularly preferred payloads are active substances and reporter molecules, especially active substances.

[0179] The term "active substance" as used in this document refers to pharmacological and / or biological substances, that is, substances that have biological and / or pharmaceutical activity, such as drugs, prodrugs, cytotoxins, diagnostic agents, proteins, peptides, polypeptides, peptide tags, amino acids, polysaccharides, lipids, vitamins, steroids, nucleotides, nucleosides, polynucleotides, RNA, or DNA. Examples of peptide tags include cell-penetrating peptides, such as human lactoferrin or polyarginine. An example of a polysaccharide is mannan oligosaccharide. An example of an amino acid is lysine.

[0180] When the payload is an active substance, the active substance is preferably selected from the group consisting of a drug and a prodrug. More preferably, the active substance is selected from the group consisting of pharmaceutically active compounds, particularly compounds with low to medium molecular weights (e.g., about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In another preferred embodiment, the active substance is selected from the group consisting of cytotoxins, antiviral agents, antibacterial agents, peptides, and oligonucleotides.

[0181] Preferred cytotoxins are selected from the group consisting of nitrogen mustard, nitrosourea, alkyl sulfonates, triazine, platinum-containing compounds, plant alkaloids, DNA topoisomerase inhibitors, antimetabolites, hormone therapy, kinase inhibitors, antibiotics, and other cytotoxins as defined below.

[0182] Suitable nitrogen mustards include chlorambucil, naphthyl mustard, cyclophosphamide, dacarbazine, estradiol, ifosfamide, nitrogen mustard, oxynitric acid mustard, mannitol mustard, dibromomannitol, melphalan, dibromoceryl alcohol, piperobromane, neonitric acid mustard, benzyl mustard cholesterol, prednimustine, thiotepa, trefophos, uramustine; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); duocarmycin (including synthetic analogs, KW-2189 and CBI-TMI); benzodiazepines. Monomers or dimers (e.g., pyrrolobenzodiazepines) (PBD), thromycin, indolinebenzodiazepine Isoindoline benzodiazepine Imidazolidine benzothiazazo and oxazolidinylbenzothiazazo ).

[0183] Suitable nitrosoureas include carmustine, lomustine, chlorhexidine, formustine, nimustine, and ramustine.

[0184] Suitable alkyl sulfonates include busulfan, trioxysulfan, indomethacin, and piperosuva.

[0185] Suitable triazines include dacarbazine.

[0186] Suitable platinum-containing compounds include carboplatin, cisplatin, and oxaliplatin; aziridines, such as benzodopa, carbaquinone, meturedopa, and uredopa; and ethyleneimines and methylamelamines, including hexamethylmelamine, tratamine, trietylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine.

[0187] Suitable plant alkaloids include vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, norvinca alkaloid), toxoids (e.g., paclitaxel, docetaxel and their analogues), maytansine alkaloids and their analogues (e.g., DM1, DM2, DM3, DM4, maytansine and anserine), cryptophytic acid (especially cryptophytic acid 1 and cryptophytic acid 8), epothilone, eleutherobin, spongilin, bryophyte lactone, dolasstatin, auristatin, tubulolysin, cefostatin, hymenopterin, styraxol and spongiformin.

[0188] Suitable DNA topoisomerase inhibitors include any camptothecin, including 9-aminocamptothecin, eczetidine, DXd (DX-8951 derivative), Krestor, donomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, nofarutamide, retinoid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000); mitomycin and mitomycin C.

[0189] Suitable antimetabolites include antifolate, DHFR inhibitors (e.g., methotrexate, trimethoprim, folate, pteroxetine, aminopterin (4-aminopterinic acid) or other folic acid analogs), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, thiazolidinedione, ribavirin, EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine), and pyrimidine analogs (e.g., uracil analogs such as ancitabine, azacitidine, 6-azapine). Urate, capecitabine (Xeloda), carmoflu, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, 5-fluorouracil, fluorouridine, raltitrexed (Tomudex); cytosine analogs (e.g., cytarabine, cytosine cytarabine, fludarabine); purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thioguanine); folic acid supplements (e.g., folic acid).

[0190] Suitable hormone therapies include receptor antagonists (e.g., anti-estrogens such as megestrol acetate, raloxifene, tamoxifen; LHRH agonists such as goscrclin, leuprorelin acetate; anti-androgens such as bicalutamide, flutamide, calotestosterone, drotaldone propionate, cyclothionol, goserelin, leuprorelin, meandrazan, nilumet, testosterone, trolostan and other androgen inhibitors), retinoids / deltoids (e.g., vitamin D3 analogs such as CB1093, EB 1089KH 1060, cholecalciferol, ergocalciferol), and photodynamic therapy (e.g., verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-rubigin A).

[0191] Suitable kinase inhibitors include BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, pilgatanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), lilotinib, panatinib (AP24534), baflutinib (INNO-406), bosutinib (SKI-606), cabozantinib, vemodega, inipatinib, ruxolitinib, CYT387, axitinib, tevozanib, sorafenib, and ispinib.

[0192] Suitable antibiotics include enediyne antibiotics (e.g., kazimycin, especially kazimycin γ1, δ1, α1, and β1), danendomycin (e.g., danendomycin A and deoxydanendomycin), esporamycin, caproicin, C-1027, maduratide, and new carcinogen chromophores and related chromogenin enediyne antibiotic chromophores, aclarubicin, actinomycin, atrazomycin, azaserine, bleomycin, actinomycin C, kanamycin, erythromycin, carcinogen; chromomycin, actinomycin D, daunorubicin (daun... (orubicin), nemorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin, epirubicin, isopycin, idarubicin, methacycline, nitromycin, mycophenolic acid, nopramine, olivomycin, pelomycin, polomycin, doxorubicin, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenmex, fenestrated statin and zorubicin.

[0193] Suitable polyketides include polyacetyl groups (especially blatasine and blatasine ketone), gemcitabine, cyclooxygenase (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedodexide, zebrestat, PLX4032, STA-9090, Stimuvax, alovitin-7, Xegeva, and Provenge.

[0194] Cytotoxins can be further selected from isoprenoidation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D and cytotoxicin), bleomycins (e.g., bleomycin A2, bleomycin B2, pilemycin), anthracyclines (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), and Ca2+ ATPase inhibitors (e.g., ... Toxic carotenoids), histone deacetylase inhibitors (e.g., vorinostat, romidesin, pabistal, valproic acid, mosetinofetide (MGCD0103), belistat, PCI-24781, entinofetide, SB939, renostat, gilvesta-stat, AR-42, CUDC-101, sulforaphane, trachomatis A), toxic carotenoids, celecoxib, glitazones, epigallocatechin gallate, disulfiram, halostamylamine A; anti-adrenergics (e.g., aminoglutethimide, mitotane, tralostertan; aceglucuronolactone; aldophosphamide glycosides). glycoside; aminolevulinic acid; acridine; cytarabine, amustine; bisaccharin; edatraxate; defofamine; demecolcine; diaziquone; DFMO, effluridine; edetrazol, etogluconol; gallium nitrate; cytosine, hydroxyurea; ibandronic acid, lentinan; chlordamine; mitoguazone; mitoantrone; mopiperazine; nitroamine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine; Razoxane; Rhizomycin; Cizonan; Germonospiramine; Tenuazonic acid; Triaminequinone; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Verrucarin A, Roridin A, and Anguidine).

[0195] The term "reporter molecule" in this article refers to a molecule whose presence is easily detectable, such as diagnostic agents, dyes, fluorophores, radiolabeled isotopes, contrast agents, magnetic resonance imaging agents, or mass markers.

[0196] Various fluorophores, also known as fluorescent probes, are known to those skilled in the art. Several fluorophores are described in more detail, for example, in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013, Chapter 10: “Fluorescent probes”, pp. 395-463 (which are incorporated herein by reference). Examples of fluorophores include all types of Alexa Fluor (e.g., Alexa Fluor 555), anthocyanin dyes (e.g., Cy3 or Cy5) and anthocyanin derivatives, coumarin derivatives, luciferin and luciferin derivatives, rhodamine and rhodamine derivatives, boron dipyrrole derivatives, pyrene derivatives, naphthylimide derivatives, phycobiliprotein derivatives (e.g., allophycocyanin), chromomycin, lanthanide chelates, and quantum dot nanocrystals.

[0197] Examples of radioactive isotope labeling include 99m Tc, 111 In、 114m In、 115 In、 18 F, 14 C 64 Cu、 131 I, 125 I, 123 I, 212 Bi、 88 Y、 90 Y、 67 Cu、 186 Rh、 188 Rh、 66 Ga、 67 Ga and 10 B, which optionally is derived via a chelating moiety (e.g., DTPA (diethylenetriaminepentaacetic anhydride), DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N″′-tetraacetic acid), DTTA (N 1 -(benzyl p-isothiocyanate)-diethylenetriamine-N 1 N 2 N 3 N 3-Tetraacetic acid), deferoxamine, or DFA (N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide) or HYNIC (hydrazine nicotinamide)) linkage. Isotopic labeling techniques are known to those skilled in the art and are described in more detail, for example, in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition 2013, Chapter 12: “Isotopic labelling techniques”, pp. 507-534 (which are incorporated herein by reference).

[0198] Polymers suitable for use as payload D in compounds according to the invention are known to those skilled in the art, and several examples are described in more detail, for example, in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013, Chapter 18: “PEGylation and synthetic polymer modification”, pp. 787-838 (which are incorporated herein by reference). When the payload D is a polymer, the payload D is preferably independently selected from the group consisting of: poly(ethylene glycol) (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1,q-diaminoalkane polymers (where q is the number of carbon atoms in the alkane, and preferably q is an integer in the range of 2 to 200, preferably 2 to 10), (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (e.g., dextran), poly(amino acids) (e.g., poly(L-lysine)), and poly(vinyl alcohol).

[0199] Suitable solid surfaces for use as payload D are known to those skilled in the art. Solid surfaces include, for example, functional surfaces (e.g., surfaces of nanomaterials, carbon nanotubes, fullerenes, or viral capsids), metallic surfaces (e.g., titanium, gold, silver, copper, nickel, tin, rhodium, or zinc surfaces), metallic alloy surfaces (wherein the alloy is derived from, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin, uranium, zinc, and / or zirconium), polymer surfaces (wherein the polymer is, for example, polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly(dimethylsiloxane) or polymethyl methacrylate, polyacrylamide), glass surfaces, silicone surfaces, chromatographic carrier surfaces (wherein the chromatographic carrier is, for example, silica carrier, agarose carrier, cellulose carrier, or alumina carrier), etc. When the payload D is a solid surface, it is preferred that D is independently selected from the group consisting of functional surfaces or polymer surfaces.

[0200] Hydrogels are known to those skilled in the art. A hydrogel is a water-swellable network formed by cross-linking between polymeric components. See, for example, Ashoffman, Adv. Drug Delivery Rev. 2012, 64, 18, which is incorporated herein by reference. When the payload is a hydrogel, it is preferred that the hydrogel is composed of poly(ethylene) glycol (PEG) as the polymeric base.

[0201] Suitable microparticles and nanoparticles for use as payload D are known to those skilled in the art. A variety of suitable microparticles and nanoparticles are described, for example, in G. T.Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013, Chapter 14: “Microparticles and nanoparticles”, pp. 549-587 (incorporated by reference). Microparticles or nanoparticles can be of any shape, such as spherical, rod-shaped, tubular, cubic, triangular, and conical. Preferably, the microparticles or nanoparticles are spherical. The chemical composition of the microparticles and nanoparticles can vary. When payload D is a microparticle or nanoparticle, the microparticle or nanoparticle may be, for example, polymer microparticles or nanoparticles, silica microparticles or nanoparticles, or gold microparticles or nanoparticles. When the particles are polymer microparticles or nanoparticles, the polymer is preferably polystyrene or a copolymer of styrene (e.g., a copolymer of styrene and divinylbenzene, butadiene, acrylates and / or vinyltoluene), polymethyl methacrylate (PMMA), polyvinyl toluene, poly(hydroxyethyl methacrylate (pHEMA)) or poly(ethylene glycol dimethacrylate / 2-hydroxyethyl acrylate) [poly(EDGMA / HEMA)]. Optionally, the surface of the microparticles or nanoparticles is modified, for example, by detergent modification, by graft polymerization of a secondary polymer, or by covalent attachment of another polymer or spacer portion, etc.

[0202] The payload D may also be a biomolecule. Biomolecules and preferred embodiments thereof are described in more detail below. When the payload D is a biomolecule, it is preferred that the biomolecule be selected from the group consisting of: proteins (including glycoproteins such as antibodies), polypeptides, peptides, polysaccharides, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides.

[0203] In the context of this invention, a cytotoxic payload is particularly preferred. Therefore, D is preferably a cytotoxic agent, more preferably selected from the group consisting of: colchicine, vinca alkaloids, anthracycline, camptothecin, doxorubicin, daunorubicin, taxane, chalcogenides, tubulolytics, irinotecan, inhibitory peptides, muscarinic acid, amatoxins, pyroxine, epothilone, mitomycin, compressoritine, maytansine, auripalin, enediyne, pyrrolobenzodiazepines. (PBD) or indolinebenzodiazepine Dimer (IGN). This invention is particularly suitable for cytotoxic payloads with moderate cytotoxicity. Typically, conjugates of such payloads require administration at higher doses, but given the high DAR (high payload load) of the conjugates of this invention, even conjugates of moderately toxic payloads can be administered at ideally low doses. In other words, D is preferably a payload that can be administered at moderate to high doses (3-20 mg / kg) in patients even at high DAR (DAR>>6). Given the high payload load in the conjugates of this invention, the mean cytotoxicity (IC50) of the payload (unconjugated form) is particularly preferred. 50 >50 nM. Therefore, even payloads with low cytotoxicity can be used in antibody-drug conjugates with high efficacy. Preferred connector L A

[0204] Especially preferred connector L A With structure - (L 11 )-BM(L 12 -)(L 13 -), where L 11 It is based on any one of (i)-(x): (i)L 11 It is *-C(O)-(CH2) a’ -C(O)-**, where the key marked as * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 1-5, preferably a' = 3. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (ii)L 11 It is *-NH-C(O)-(CH2) a’ -**, where the key marked as * is related to Q 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 0-5, preferably a' = 0. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (iii)L 11 It is *=NC(O)-(CH2) a’ -**, where the double bond marked as * is related to Q. 1 or Z 1The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 0-5, preferably a' = 0. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (iv)L 11 It is *-NH-C(O)-(CH2) a’ -C(O)-**, where the key marked as * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 1-5, preferably a' = 3. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (v)L 11 It is *=NC(O)-(CH2) a’ -C(O)-**, where the double bond marked as * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 1-5, preferably a' = 3. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (vi)L 11 It is *-C(O)-**, where the bond marked as * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, wherein preferably BM is a nitrogen atom (for y=2) or a carbon atom (for y=3). Most preferably, BM=N. (vii)L 11 It is *-(23)-(CH2CH2O) a’ -(C(O)) a” -**, where the key marked as * is related to Q 1 or Z 1 The bond is connected to BM, and the bond marked as ** is connected to BM, where (23) refers to the group according to structure (23). Additionally, a' is an integer in the range of 0-5, preferably a' = 1-4, most preferably a' = 2, and a” is 0 or 1, preferably a” is 1. Furthermore, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (viii)L 11 It is *-(23)-(CH2CH2O) a’ -(23)-**, where the key marked as * is related to Q 1 or Z 1The bond is connected to BM, and the bond marked with ** is connected to BM, where (23) refers to the group according to structure (23). Additionally, a' is an integer in the range of 0-5, preferably a' = 1-4, and most preferably a' = 2. Furthermore, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (ix)L 11 It is *-C(O)-NH-(CH2CH2O) a’ -C(O)-**, where the key marked as * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 1-5, preferably a' = 3. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N. (x)L 11 It is *-C(O)-NH-(CH2CH2O) a’ -OC(O)-NH-**, where the bond marked with * is related to Q. 1 or Z 1 The bond is connected to BM, and the bond marked with ** is connected to BM, where a' is an integer in the range of 1-5, preferably a' = 3. Additionally, it is preferred that BM is a nitrogen atom (for y = 2) or a carbon atom (for y = 3). Most preferably, BM = N.

[0205] Especially preferred connector L A With structure - (L 11 )-BM(L 12 -)(L 13 -), where L 12 and L 13 It is based on any one of (xi)-(xv): (xi)L 12 and L 13 Both are *-(CH2) a’ -OC(O)-NH)-(CH2CH2O) a” -CH2-CH2-NC(O)-**, where the bond marked * is connected to BM, and the bond marked ** is connected to F. 2 The connection is defined as follows: a' is an integer in the range of 0-4, preferably a' = 2, and a” is an integer in the range of 0-4, preferably a” = 2. Additionally, BM is preferably a nitrogen atom. (xii)L 12 and L 13 Both are *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a”-Ph-**, where the key marked * is concatenated with BM, and the key marked ** is concatenated with F. 2 The connection is defined as follows: a' is an integer in the range of 0-5, preferably a' = 2, and a” is an integer in the range of 0-4, preferably a” = 1. Preferably, Ph is 1,4-Ph. Additionally, preferably, BM is a nitrogen atom. (xiii)L 12 and L 13 Both are *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a” -NH-C(O)-Ph-**, where the bond marked * is connected to BM, and the bond marked ** is connected to F. 2 The connection is defined as follows: a' is an integer in the range of 0-5, preferably a' = 2, and a” is an integer in the range of 0-4, preferably a” is 2 or 3. Preferably, Ph is 1,4-Ph. Additionally, preferably, BM is a nitrogen atom. (xiv)L 12 and L 13 Both are *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a” -O-Ph-**, where the key marked * is concatenated with BM, and the key marked ** is concatenated with F. 2 The connection is defined as follows: a' is an integer in the range of 0-5, preferably a' = 2, and a” is an integer in the range of 0-4, preferably a” is 2 or 3. Preferably, Ph is 1,4-Ph. Additionally, preferably, BM is a nitrogen atom. (xv)L 12 and L 13 Both have a structure-(L 14 )-BM'(L 15 -)(L 16 -),in: -L 14 Is this for L? 11 The definition is preferably based on any one of (i) to (x) as defined above, or L 14 It is *-(CH2CH2O) a’ -CH2-CH2-(C(O)) a” -**, where the key marked as * is connected to BM, and the key marked as ** is connected to BM', and where a' is an integer in the range of 0-5, preferably a' = 1-4, most preferably a' = 2, and a” is 0 or 1, preferably a” is 1. -BM and BM' are branching portions, preferably both being nitrogen atoms. In a single occurrence of BM and a double occurrence of BM' (L...15 and L 16 In the case of each of the following, y = 4. -L 15 and L 16 For example, regarding L 12 and L 13 Defined. Preferably, according to the definition above (xiv).

[0206] Preferably, connector L 12 and L 13 Both have the same structure. In L A In a particularly preferred embodiment, connector L 11 It is based on any one of (i)-(x), and connects the sub-L 12 and L 13 It is based on any one of (xi)-(xv).

[0207] Such a preferred connector L A It can be used in the linker-construction according to the invention, the conjugate according to the invention, and the intermediate antibody-linker construct according to the invention. In the context of this preferred embodiment, Q 1 Preferably, it is based on (Q26) or (Q37), and F 2 Preferably, it is based on (F8a), where R 29 It is hydrogen or methyl. Alternatively, Z is preferred. 1 Preferably, it is based on (Z26) or (Z37), wherein ring Z is based on (Za), and Z 2 Preferably, it is based on (Z29), where ring Z is based on (Zj), where R 29 It is either hydrogen or methyl. Synthesis method of couplings based on general structure (2)

[0208] In another aspect, the present invention relates to a method for preparing a coupling compound according to the invention, the method comprising: (a) Provides a structure Ab(F) 1 ) z The modified antibody, where Ab is an antibody, z is 2 or 4, and F 1 It is a click probe; (b) Mix the modified antibody with z equivalents of Q 1 L A (F 2 ) y The reaction, in which Q 1 It is for F 1 Reactive click probe, L A It is a heterofunctional (y+1) valence linker, where y is 2, 3, or 4, and F 2 It is for Q1 Non-reactive click probes to obtain structures with Ab(Z) 1 L A (F 2 ) y ) z The antibody-linker construct, in which Z 1 Through F 1 and Q 1 Linking groups obtained from the reaction; (c) Make the antibody-linker construct with a z×y equivalent of Q 2 (L B )D x The reaction, in which Q 2 It is for F 2 Reactive click probe, L B It is a (x+1) valence linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is the effective load molecule, to obtain the structure Ab(Z). 1 L A (Z 2 (L B )D x ) y ) z Coupled with Z 2 Through F 2 and Q 2 Linking groups obtained from the reaction.

[0209] The reactions performed in steps (b) and (c) are click reactions, in which click probe F reacts with click probe Q to form linker group Z. This coupling technique is known to those skilled in the art. This reaction occurs under conditions where Q reacts with F to form a covalent link. In the method according to the invention, Q reacts with F, thereby forming a covalent link between the antibody and the payload. Complementary reactive groups Q and F are known to those skilled in the art and are described in more detail below.

[0210] Therefore, in the reaction according to the method of the invention, coupling is accomplished via cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reactions) or [3+2]-cycloadditions (e.g., 1,3-dipolar cycloadditions). The reaction in step (b) is preferably a [3+2]-cycloaddition, more preferably a 1,3-dipolar cycloaddition. The 1,3-dipolar cycloaddition is preferably an alkyne-azide cycloaddition, and most preferably wherein Q is or contains an alkyne group and F is an azide group. The reaction in step (c) is a [4+2]-cycloaddition, preferably a Diels-Alder reaction. Preferred Diels-Alder reactions are reverse electron-demanding Diels-Alder cycloadditions. Cycloadditions (such as Diels-Alder reactions and 1,3-dipolar cycloadditions) are known in the art, and those skilled in the art know how to perform them. Step (a)

[0211] In step (a), a structure Ab(F) is provided. 1 ) z Antibodies. Typically, two or four click probes F 1 The probes are introduced onto the antibody chemically or enzymatically. Preferably, z = 2, and two click probes are introduced onto the antibody.

[0212] In a preferred embodiment, an antibody comprising two or four (preferably two) core N-acetylglucosamine moieties is reacted with an antibody having the formula S(F 1 Compounds of )-P are contacted in the presence of a catalyst, wherein S(F 1 It contains two reactive groups Q. 1 The reactive group F in the reaction 1 The sugar derivative, wherein P is a nucleoside monophosphate or nucleoside diphosphate, and wherein the catalyst is capable of converting S(F) into a phosphate group. 1 The antibody is partially transferred to the core-GlcNAc region. In this paper, the antibody is typically an antibody that has been pruned to the core-GlcNAc residues, as further described below.

[0213] Starting materials, i.e., antibodies containing two or four, preferably two, core-GlcNAc substituents, are known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the method according to the invention further comprises deglycosylation of an antibody glycan having a core N-acetylglucosamine substituent in the presence of a glycosidase to obtain an antibody containing a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. A suitable glycosidase can be selected according to the properties of the glycan. The glycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH and / or combinations thereof, the selection depending on the properties of the glycan. EndoSH is described in PCT / EP2017 / 052792, see Examples 1-3 and SEQ.ID No:1, which is incorporated herein by reference.

[0214] The structural feature S, as defined above for the coupling according to the invention, is equally applicable to this aspect. It has the formula S(F) 1 Compounds of )-P are known in the art, wherein nucleoside monophosphate or nucleoside diphosphate P is reacted with sugar derivative S(F) 1 For example, Wang et al., Chem. Eur. J. [Chemistry-European Journal] 2010, 16, 13343-13345; Piller et al., ACS Chem. Biol. [ACS Chemical Biology] 2012, 7, 753; Piller et al., Bioorg. Med. Chem. Lett. [Bioorganic and Medicinal Chemistry Letters] 2005, 15, 5459-5462 and WO 2009 / 102820 (all of which are incorporated herein by reference) disclose numerous compounds S(F 1 S(F)-P and its synthesis. In a preferred embodiment, S(F) 1 The nucleoside monophosphate or nucleoside 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 1S(F)-P is selected from the group consisting of: GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetylgalactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP, and 2-(but-3-acetylamido)-2-deoxy-galactose-UDP. Most preferably, S(F) 1 -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0215] Able to convert S(F) 1 Suitable catalysts for the transfer of the nucleotide S(F) to the core-GlcNAc moiety are known in the art. Suitable catalysts are those that enable the transfer of a specific sugar derivative nucleotide S(F) to the core-GlcNAc moiety in this particular method. 1 The catalyst uses β-P as a substrate. More particularly, the catalyst catalyzes the formation of β(1,4)-glycosidic bonds. Preferably, the catalyst is selected from the group consisting of galactosyltransferases and N-acetylgalactosaminetransferases, more preferably from the group consisting of β(1,4)-N-acetylgalactosaminetransferase (GalNAcT) and β(1,4)-galactosyltransferase (GalT), and most preferably from the group consisting of β(1,4)-N-acetylgalactosaminetransferases having a mutant catalytic domain. Suitable catalysts and their mutants are disclosed in WO 2014 / 065661, WO 2016 / 022027 and WO 2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or an N-acetylgalactosaminetransferase, preferably an N-acetylgalactosaminetransferase. In alternative embodiments, the catalyst is a mutant galactosyltransferase or N-acetylgalactosamine transferase, preferably a mutant N-acetylgalactosamine transferase. The mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts are capable of recognizing internal sugars and sugar derivatives as acceptors. Therefore, sugar derivatives S(F 1 In step (a), it is linked to the core-GlcNAc substituent, regardless of whether the GlcNAc is fucosylated.

[0216] Step (a) 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 buffer solutions 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°C to about 50°C, more preferably in the range of about 10°C to about 45°C, even more preferably in the range of about 20°C to about 40°C, and most preferably in the range of about 30°C to about 37°C. Step (i) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (i) is carried out at a pH in the range of about 7 to about 8. Step (b)

[0217] In step (b), the modified antibody Ab(F) 1 ) z Reaction with the linker construct according to the invention, the linker construct comprising a reactive group F 1 The reactive group Q of the reaction 1 To obtain an antibody-linker construct containing Q 1 With F 1 The connecting group Z produced by the reaction between them 1 Such a reaction occurs under the following conditions: the reactive group Q... 1 The reactive group F of the antibody 1 The reaction is carried out to covalently link the antibody to the linker-construct. In step (b), the reaction is carried out at z equivalents of Q. 1 L A (F 2 ) y This occurs under certain circumstances, despite more equivalent Q. 1 L A (F 2 ) y It can be present in the reaction mixture to ensure complete reaction. Technicians are able to determine the optimal reaction conditions and stoichiometry of the reactants to obtain the best yield.

[0218] In a preferred embodiment, in step (b), the azide on the azide-modified antibody reacts with a benzo[i]cyclocyclized or tetramethylated (hetero]cycloalkyne group via a cycloaddition reaction, preferably wherein Q 1The structures are (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a), preferably (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a), more preferably (Q40), (Q41), or (Q43), or (Q37) or (Q43). Using such benzo-cyclized or tetramethylated (hetero)cyclic alkyne groups, the presence of a catalyst is not required, and the cycloaddition reaction can even occur spontaneously via a reaction known as strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions referred to in the art as "metal-free click chemistry." Step (c)

[0219] In step (c), the modified antibody-linker construct Ab(Z) 1 L A (F 2 ) y ) z Reaction with payload-linker, the payload-linker comprising a reactive group F 2 The reactive group Q of the reaction 2 To obtain an antibody-conjugate containing Q 2 With F 2 The connecting group Z produced by the reaction between them 2 Such a reaction occurs under the following conditions: the reactive group Q... 2 With reactive group F 2 The reaction covalently links the antibody to the payload. Step (c) can also be referred to as the coupling reaction. In step (c), the reaction occurs at a z×y equivalent of Q. 2 (L B )D x This occurs under certain circumstances, despite more equivalent Q. 2 (L B )D x It can be present in the reaction mixture to ensure complete reaction. Technicians are able to determine the optimal reaction conditions and stoichiometry of the reactants to obtain the best yield.

[0220] In step (c), the conjugate is formed by covalently linking multiple payloads to the antibody. In this step, the DAR of the conjugate is established. The conjugation step of the present invention is particularly effective because it provides conjugates with an average DAR close to the theoretical value. Therefore, the conjugates according to the present invention exhibit high homogeneity. The inventors further discovered that on the antibody side of the conjugation reaction, i.e., at Ab(Z) 1 L A (F2 ) y ) z In, usually in L A The negative charge within the antibody-linker negatively impacts the coupling reaction, potentially resulting in lower DAR values. However, the method according to the invention enables the processing of such negatively charged modified antibody-linker constructs, yielding conjugates with unprecedented DAR values. A The negative charge in the process is present under the method conditions of step (c) and can, for example, originate from the deprotonation of a carboxylic acid group or a sulfonamide group (e.g., according to structure (23)). Therefore, in one embodiment, the linker L... A It is non-negatively charged and preferably does not contain carboxylic acid groups or sulfonamide groups. In alternative embodiments, the linker L A It is negatively charged and preferably contains carboxylic acid groups and / or sulfonamide groups.

[0221] In a preferred embodiment, in step (c), the 1,2,4,5-tetraazine on the antibody-linker construct reacts with the bicyclic nonyne group via a cycloaddition reaction, preferably wherein Q 1 It is according to structure (Q8), more preferably according to structure (Q29), and most preferably according to structure (Q42). Using such a bicyclic nonyne group, the presence of a catalyst is not required, and the cycloaddition reaction can even occur spontaneously via a reaction known as strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions referred to in the art as "metal-free click chemistry." Reaction part Q

[0222] Reaction part Q 1 and Q 2 It is a click probe. In the context of this invention, Q refers to Q. 1 and Q 2 In the context of this invention, the term "reactive moiety" can refer to the chemical moiety containing the reactive group or the reactive group itself. For example, a cyclooctyne group is a reactive group containing a reactive group, namely a C-C triple bond. However, a reactive group (e.g., an azide reactive group) may also be referred to herein as a reactive moiety.

[0223] Q is reactive with and complementary to F. In this document, a reactive group is referred to as being “complementary” to another reactive group when the reactive group selectively reacts with it (optionally in the presence of other functional groups). Complementary reactive click probes are known to those skilled in the art and are described in more detail below. The exact properties of Q and F depend on the type of click reaction employed. Click probes are reactive in cycloaddition (click reactions) and are preferably selected from azides, tetrazides, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, sine ketones, olefinic moieties, and alkyne moieties. Preferably, the click probe Q comprises either an olefinic moiety or an alkyne moiety, more preferably wherein the olefin is a (hetero)cyclic olefin and / or the alkyne is a terminal alkyne or a (hetero)cyclic alkyne.

[0224] In a particularly preferred embodiment, Q comprises a cyclic (hetero)yne moiety. The ynyl group may also be referred to as a (hetero)cyclic ynyl group, i.e., a heterocyclic ynyl group or a cycloynyl group, wherein the (hetero)cyclic ynyl group is optionally substituted. Preferably, the (hetero)cyclic ynyl group is a (hetero)cyclic heptyynyl group, a (hetero)cyclic octyynyl group, a (hetero)cyclic nonynyl group, or a (hetero)cyclic decanynyl group. In this document, the (hetero)cyclic ynylene may optionally be substituted. Preferably, the (hetero)cyclic ynyl group is an optionally substituted (hetero)cyclic heptyynyl group or an optionally substituted (hetero)cyclic octyynyl group. Most preferably, the (hetero)cyclic ynyl group is a (hetero)cyclic octyynyl group, wherein the (hetero)cyclic octyynyl group is optionally substituted.

[0225] In a particularly preferred embodiment, Q comprises a (hetero)cyclic alkynyl or (hetero)cyclic alkenyl group and is according to structure (Q1): In this article: - Described as The key is a double or triple bond; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -Y 2 It is C(R) 31 )2、O、S、S (+) R 31 S(O)R 31 S(O) = NR 31 or NR 31 S (+) Is with B (-) Mutually balanced cation sulfur atoms, of which B (-) It is an anion, and each R in it is an anion. 31 R alone 15 Or a connection to D (via L); -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u+u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; -v = integers in the range 0-16;

[0226] Typically, v = (u + u') x 2 (when the connection with L (described by a tilde) is via Y) 2 (when) or [(u+u')x 2]-1 (when the connection with L (described by a tilde) is via one of the carbon atoms in u and u').

[0227] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cyclic alkynyl group and is according to structure (Q1a): In this article, -R 15 and Y 2 As defined above; -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u+u' = 4, 5, 6, 7 or 8; -v = integers in the range of 8-16.

[0228] In a preferred embodiment, u+u' = 4, 5 or 6, more preferably u+u' = 5.

[0229] In a preferred embodiment, v = 8, 9 or 10, more preferably v = 9 or 10, and most preferably v = 10.

[0230] In a preferred embodiment, Q is a (hetero)cyclic alkynyl group selected from the group consisting of (Q2)-(Q20) described below.

[0231] In this paper, the connection with L (described by a tilde) can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) can be connected to L, or can contain hydrogen atoms or be optionally functionalized. (-) It is an anion, preferably selected from... (-) OTf, Cl (-) ,Br (-) Or I (-) The most preferred option is B. (-) yes (-) OTf. B (+) It is a cation, preferably a pharmaceutically acceptable cation. In the coupling reaction, B (-) It does not need to be a pharmaceutically acceptable anion, because B (-) In any case, it will exchange with the anions present in the reaction mixture. If (Q19) is used as Q, then the negatively charged counter-charged ion is preferably pharmaceutically acceptable when separating the conjugate according to the invention, so that the conjugate can be readily used as a pharmaceutical agent.

[0232] In another preferred embodiment, Q is a (hetero)cyclic alkynyl group selected from the group consisting of (Q21)-(Q38a) described below.

[0233] In structure (Q38), B (-) It is an anion, preferably selected from... (-) OTf, Cl (-) ,Br (-) Or I (-) The most preferred option is B. (-) yes (-) OTf.

[0234] In a preferred embodiment, Q comprises a (hetero)cyclic octynylene moiety or a (hetero)cyclic heptaynylene moiety, preferably according to structures (Q8), (Q26), (Q27), (Q28), (Q37), or (Q38a), which may optionally be substituted. Each of these preferred options for Q is further defined below.

[0235] Therefore, in a preferred embodiment, Q comprises the heterocyclic heptyne moiety according to structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocyclic heptyne moiety according to structure (Q37) is not substituted.

[0236] In an alternative preferred embodiment, Q comprises a cyclooctynyl moiety according to structure (Q8), more preferably according to (Q29), also referred to as a bicyclic [6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctynyl moiety according to structure (Q8) or (Q29) is not substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctynyl moiety according to structure (Q39) as shown below, wherein V is (CH2). l And l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, even more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q is further defined below according to structure (Q42).

[0237] In alternative preferred embodiments, Q comprises a (hetero)cyclic octylene moiety according to structure (Q26), (Q27), or (Q28), also referred to as a DIBO, DIBAC, DBCO, or ADIBO group, which may optionally be substituted. In the context of this embodiment, Q is preferably a (hetero)cyclic octylene moiety according to structure (Q40) or (Q41), as shown below, wherein Y 1 Is it O or NR? 11 , where R 11 Independently selected from the group consisting of: hydrogen, straight-chain or branched C1-C 12 alkyl groups or C4-C 12 (Hetero)aryl groups. The aromatic ring in (Q40) is optionally O-sulfonated at one or more positions, while the ring in (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclic octylene moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), as further defined below.

[0238] In an alternative preferred embodiment, Q comprises a heterocyclic heptynyl group and is according to structure (Q37).

[0239] In a particularly preferred embodiment, Q comprises a cyclooctyne group and is according to structure (Q42): In this article: -R 15Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C5-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -R 18 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -R 19 Choose from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, which are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N, and S, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are independently and optionally substituted, or R 19 It is the second occurrence of Q or D connected via a gap; and -l is an integer in the range of 0 to 10.

[0240] In a preferred embodiment of the reactive group according to structure (Q42), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R16 It is hydrogen or C1-C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably all R 15 All are H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 Independently selected from the group consisting of: hydrogen, C1-C6 alkyl groups, most preferably two R groups. 18 All are H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 It is H. In a preferred embodiment of the reactive group according to structure (Q42), l is 0 or 1, more preferably l is 1.

[0241] In a particularly preferred embodiment, Q comprises a (hetero)cyclic octyryl group and is according to structure (Q43): In this article: -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl groups, C5-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 alkyl(hetero)aryl groups and C7-C 24 (Hetero)arylalkyl groups; -Y is N or CR 15 ; - The carbon atom in the fused aromatic ring can be replaced by a nitrogen atom as shown in (Q6a)-(Q6d), preferably where Y is CR 15 .

[0242] In a preferred embodiment of the reactive group according to structure (Q43), R 15Independently select from the following groups: hydrogen, halogen, -OR 16 -S(O)3 (-) C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1-C6 alkyl, more preferably R 15 Independently select from the following groups: hydrogen and -S(O)3 (-) In a preferred embodiment of the reactive group according to structure (Q43), Y is N or CH, more preferably Y = N.

[0243] In alternative preferred embodiments, Q comprises a cyclic olefin moiety. The alkenyl group Q may also be referred to as a (hetero)cyclic alkenyl group, i.e., a heterocyclic alkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cyclic alkenyl group is optionally substituted. Preferably, the (hetero)cyclic alkenyl group is a (hetero)cyclic propenyl group, a (hetero)cyclic butenyl group, a norbornenyl group, a norbornadienyl group, a trans-(hetero)cyclic heptenyl group, a trans-(hetero)cyclic octenyl group, a trans-(hetero)cyclic nonenyl group, or a trans-(hetero)cyclic decenyl group, all of which may be optionally substituted. Particularly preferred are the (hetero)cyclic propenyl group, the trans-(hetero)cyclic heptenyl group, or the trans-(hetero)cyclic octenyl group, wherein the (hetero)cyclic propenyl group, the trans-(hetero)cyclic heptenyl group, or the trans-(hetero)cyclic octenyl group is optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclic butene moiety according to structure (Q45), a norbornene or norbornediene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). In this document, Y 3 Selected from C(R) 23 2. NR 23 Or O, where each R 23 It is hydrogen alone, C1-C6 alkyl, or linked to L (optionally via a spacer), and is labeled with The bond is a single or double bond. In another preferred embodiment, the cyclopropenyl group is according to structure (Q49). In yet another preferred embodiment, the trans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In yet another preferred embodiment, the trans-(hetero)cyclooctene group is according to structure (Q52), (Q53), (Q54), (Q55), or (Q56).

[0244] In this document, the R group on Si in (Q50) and (Q51) is usually alkyl or aryl, preferably C1-C6 alkyl.

[0245] In a preferred embodiment, the click probe Q includes a portion selected from (Q1)-(Q56), more preferably a portion selected from (Q1)-(Q56).

[0246] In this invention, Q 1 and Q 2 The exact structure should be different because Q 1 For F 2 It is not reactive, while Q 2 For F 2 It is reactive.

[0247] In a preferred embodiment, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is either tetrazine or nitroacetate, and Q 2 It is a bicyclic nonyne or a cyclic olefin, such as trans-cyclooctene or cyclopropene. More preferably, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is tetrazine, and Q 2 It is a bicyclic nonyne. In this paper, Q... 1 Preferably, it is based on structures (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a); more preferably, it is based on structures (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a); and most preferably, it is based on structures (Q40), (Q41), or (Q43), or on structures (Q37) or (Q43). In this document, Q... 2 Preferably, it is based on structures (Q8), (Q44), (Q47), (Q48), (Q49), (Q54), (Q55), or (Q56), more preferably on structures (Q29), (Q48), or (Q49), and most preferably on structure (Q42). Alternatively, Q 2 Preferably, it is based on structure (Q8), more preferably on structure (Q29), and most preferably on structure (Q42). Reaction part F

[0248] Reaction part F 1 and F 2 It is a click probe. In the context of this invention, F refers to F. 1 and F 2F is reactive with and complementary to Q. In this document, a reactive group is referred to as "complementary" to another reactive group when the reactive group selectively reacts with it (optionally in the presence of other functional groups). Complementary reactive click probes are known to those skilled in the art and are described in more detail below. The exact properties of Q and F depend on the type of click reaction employed. Click probes are reactive in cycloaddition (click reactions) and are preferably selected from azides, tetrazides, triazides, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, sine ketones, olefinic moieties, and alkyne moieties. Preferably, click probe F comprises either an azide moiety, a nitrone moiety, or a tetrazide moiety.

[0249] F is reactive to Q in the coupling reaction defined below, preferably wherein the coupling reaction is a cycloaddition reaction or a nucleophilic reaction. Those skilled in the art will understand that the options for F are the same as those for Q, provided that F and Q are reactive to each other. The click probe is reactive in the cycloaddition (click reaction) and is preferably selected from azides, tetrazides, triazides, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, syringones, olefinic moieties, and alkyne moieties. Preferably, the click probe comprises an azide, tetrazide, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o-quinone, dioxothiophene, or syringone, most preferably an azide.

[0250] The reactive group F on an antibody is typically introduced using specific techniques (e.g., (bio)chemical or genetic techniques). The reactive group incorporated into the antibody is prepared through chemical synthesis, such as an azide or terminal alkyne. Methods for preparing modified antibodies are known in the art, for example from WO 2014 / 065661, WO 2016 / 170186, and WO 2016 / 053107, which are incorporated herein by reference. From the same literature, the coupling reaction between the modified antibody and the linker-toxin-construct is known to those skilled in the art.

[0251] Preferably, F is a click probe reactive to (hetero)cyclic alkenes and / or (hetero)cyclic alkynes, and is generally selected from the group consisting of: azides, tetrazides, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, and syl ketones. The preferred structures of the reactive group are those described below (F1)-(F10).

[0252] In this article, the tilde key represents an abbreviation with Ab or L. A The payload can be attached to any one of the tilde bonds for (F3), (F4), (F8), and (F9). Other tilde bonds can then be attached to R groups selected from: hydrogen, C1-C...24 Alkyl groups, C2-C 24 Acyl group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 The sulfonyl groups, each of which (except hydrogen) may optionally be substituted and optionally selected from O, S, and NR. 32 One or more heteroatoms are interrupted, where R 32 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 groups can be applied to each group F. For example, the R group attached to the nitrogen atom in (F3) can be selected from alkyl and aryl groups, while the R group attached to the carbon atom in (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl groups. Preferably, the reactive moiety F is selected from azides or tetrazines.

[0253] In a particularly preferred embodiment, F is a tetrazine according to structure (F8a):

[0254] In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction is optimal. Therefore, in preferred embodiments, the cyclic F (especially F2) produces the best results. 2 ) is (F8a), where R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen or methyl, most preferably R 29 It is a methyl group.

[0255] In a preferred embodiment, click probe F 1 and F 2The group consisting of the following is selected: azides, tetrazides, triazines, nitrones, nitriles, nitriles, nitrile imines, diazo compounds, dioxothiophene, syringones, iminosyringones, and catechols. It should be noted that catechols are oxidized in situ to an ortho-quinone group, which is reactive as a click probe. Similarly, the term "tetrazine" also encompasses "hydrotetrazine," a known precursor that forms a tetrazine upon in situ oxidation. This invention also covers such precursors for click probes that form reactive groups in situ. Figure 11 Some known examples of click probes formed in situ through oxidation are given. A preferred click probe F... 1 and F 2 The azide is selected from the group consisting of: azides, tetrazides, o-quinones, and nitrones, more preferably selected from azides, tetrazides, and nitrones. In a preferred embodiment, F 1 It is an azide or nitroketone, and F 2 It is an iminosine, catechol (forming an ortho-quinone group in situ), or tetraazine. More preferably, F 2 It is an iminosine based on structure (F7), a catechol (forming structure (F10) in situ), or a tetrazine based on structure (F8). Even more preferably, F 1 It is an azide according to structure (F1) or a nitroketone according to structure (F3), and F 2 It is a tetrazine based on the structure (F8a). Most preferably, F... 1 It is an azide based on the structure (F1), and F 2 It is a tetrazine based on the structure (F8a). Connector-Constructor Based on General Structure (4)

[0256] In another aspect, the present invention relates to linker-constructors. The linker-constructor according to the invention is a heterobifunctional (y+1) valence linker having structure (4): Q 1 L A (F 2 ) y (4) In this article, Q 1 It's a click probe, L A It is a connector, y is 2, 3 or 4, and F 2 It is for Q 1 Non-reactive click probes. Q 1 L A y and F 2 As defined elsewhere, and those definitions and their preferred embodiments also apply to connector-constructors according to the invention.

[0257] In a preferred embodiment of the connector-constructor according to this aspect, Q 1It is a benzo[a]cyclic or tetramethylated (hetero]cyclic alkyne. Such a click probe Q 1 Preferably with F as a click probe 2 The tetrazine combination. Therefore, Q 1 and F 2 Both are reactive click probes in cycloaddition reactions, but they are not reactive with each other.

[0258] Q 1 Preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), and (Q19a) as defined above, more preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q18), and (Q19a), even more preferably according to structures (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a), and most preferably according to structures (Q37), (Q40), (Q41), or (Q43).

[0259] F 2 Preferably, it is a tetrazine based on structure (F8), and most preferably, it is based on structure (F8a): In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl, preferably wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. In a preferred embodiment, R 33 It is hydrogen or methyl, most preferably R 33 It is a methyl group.

[0260] The integer y is 2, 3 or 4, preferably y is 2 or 3, and most preferably y is 2. application

[0261] The conjugates of the present invention with a DAR of 6 or higher exhibit high homogeneity and a DAR value close to the theoretical DAR value. The conjugates of the present invention are further characterized by high stability, low aggregation tendency, and excellent therapeutic efficacy and tolerability. Therefore, the conjugates of the present invention are particularly suitable for treating cancer. In view of this, the present invention further relates to a method of treating cancer, comprising administering the conjugate according to the present invention to a subject in need. The subject in need is typically a cancer patient. The use of conjugates (such as antibody-drug conjugates) is well known in the field of cancer treatment, and the conjugates according to the present invention are particularly suitable for this purpose. The described method is generally suitable for treating cancer. In the method according to this aspect, the antibody-drug conjugate is typically administered at a therapeutically effective dose. This aspect of the present invention can also be expressed as the use of the conjugates according to the present invention in the treatment of cancer. In other words, this aspect relates to the use of the conjugates according to the present invention in the preparation of pharmaceutical agents or pharmaceutical compositions for use in the treatment of cancer. In this context, cancer treatment is considered to encompass treating tumors, imaging tumors, diagnosing tumors, preventing tumor proliferation, preventing tumor spread (containing tumor), and reducing tumor size.

[0262] This aspect of the invention can also be described as a method for targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting a conjugate according to the invention with cells that may express the extracellular receptor, wherein an antibody specifically targets the extracellular receptor. Therefore, the method according to this aspect is suitable for determining whether cells express a desired extracellular receptor. These tumor cells may be present in a subject, in which case the method comprises administering the conjugate according to the invention to the subject in need. Alternatively, the method may occur in vitro or ex vivo. In a preferred embodiment, the cells that may express the extracellular receptor are cells that express the extracellular receptor. Targeting tumor cells preferably includes treating tumor cells, imaging tumor cells, diagnosing tumor cells, preventing tumor cell proliferation, preventing tumor cell spread (containing tumor cells), and reducing one or more types of tumor cells.

[0263] In the context of diagnosis, it is often unknown whether the cells being treated actually express the specific extracellular receptor being investigated. For example, in the diagnosis of HER2-positive breast cancer, a conjugate containing an antibody targeting HER2 (such as trastuzumab) can be applied to the cells. If the tumor cells do express HER2, the conjugate will target those cells; if the tumor cells do not express HER2, the conjugate will not. Similarly, when treating cancer cells that specifically express an extracellular receptor, the technician will understand that a cell-binding agent (such as an antibody) targeting that specific extracellular receptor should be used.

[0264] In the method of the present invention, the extracellular receptor is preferably selected from the group consisting of: 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, short proteoglycan, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / glucan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD 38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, Sealin-18.2, Sealin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, Fibronectin, FLT3, Folate receptor α, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylate cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, connexin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tendinin. Similarly, it is preferred that tumor cells express extracellular receptors selected from the same group. Those skilled in the art can match the desired extracellular receptor with a suitable cell binder capable of targeting that extracellular receptor.

[0265] The conjugates of the present invention are also particularly suitable as antibiotics, antivirals, anti-inflammatory agents, and anti-autoimmune agents. Therefore, in alternative embodiments, the present invention relates to a method for treating infection, inflammation of autoimmune disorders, comprising administering the conjugate according to the invention to a subject in need. In the method according to this aspect, the antibody-conjugate is typically administered at a therapeutically effective dose. This aspect of the invention can also be expressed as the use of the conjugate according to the invention in treating infection, inflammation of autoimmune disorders. In other words, this aspect relates to the use of the conjugate according to the invention in the preparation of pharmaceutical agents or pharmaceutical compositions for use in treating infection, inflammation of autoimmune disorders. In this context, infection can be bacterial or viral.

[0266] In this embodiment, the antibody is preferably specific to extracellular proteins and / or tumor-associated carbohydrate antigens caused by viral infection. In this document, extracellular proteins caused by viral infection can be human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). Tumor-associated carbohydrate antigens (TACA) can be selected from the group consisting of: Tn, STn, T antigen, LDN, Lewis antigen, etc. c (Le c ), sialic acid-Lewis c (SLe c ), 6-sialic acid-Lewis c (6SLe c ), LN, α-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis antigen a (Le a ), sialic acid-Lewis a (SLe a ), 6-sialic acid-Lewis a (6SLe a Louis b (Le b ), sialic acid-Lewis b (SLe b ), 6-sialic acid-Lewis b (6SLe b Louis x (Le x ), sialic acid-Lewis x (SLe x ), 6-sialic acid-Lewis x (6SLe x Louis y (Le y ), sialic acid-Lewis y (SLe y ), 6-sialic acid-Lewisy (6SLe y Antibodies can be specific to both extracellular proteins and TACA.

[0267] In view of this, the present invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a conjugate according to the invention. The pharmaceutical compositions typically contain a pharmaceutically effective dose of the conjugate according to the invention. Attached Figure Description

[0268] Figure 1 A general protocol for preparing antibody-drug conjugates is shown, which involves the reaction of monoclonal antibodies containing x functional groups F (most often symmetrical dimers). This is achieved by... x The conjugate is obtained by incubating with an excess of linker-drug construct (Q-spacer-linker-payload) and forming linker group Z by the reaction of F with Q.

[0269] Figure 2 A series of reagents suitable for reaction with cysteine ​​side chains are described. The reagents can be of the monoalkylation type (A) or crosslinking agents that react with two cysteine ​​side chains (B).

[0270] Figure 3 A general method for non-genetically converting monoclonal antibodies (mAbs) into antibodies containing probes (F) for click coupling is shown. The click probes can be located at different positions on the antibody, depending on the technique used. For example, the antibody can be converted into an antibody containing two click probes (left-side structure), four click probes (bottom structure), or eight probes (right-side structure) for click coupling.

[0271] Figure 4 A representative (but not exhaustive) set of functional groups (F) is shown that can be introduced into antibodies via engineering, chemical modification, or enzymatic methods, and generate linker groups Z after a metal-free click reaction with complementary reactive groups Q. Functional groups F can be artificially introduced (engineered) into any selected position on the antibody. Some functional groups F (e.g., nitrile oxides, quinones) can react with strained alkynes as well as strained alkenes, such as triazines or tetraazines (bottom row). Pyridine or pyridazine linker groups are products of rearrangement of tetraazabicyclo[2.2.2]octane linker groups formed by the reaction of triazines or tetraazines with alkynes (but not alkenes) and the loss of N2. Figure 4 The linking group Z depicted is a preferred linking group used in this invention.

[0272] Figure 5Preferred examples of cyclic alkynes suitable for metal-free click chemistry and reaction motif Q are shown. This list is not exhaustive; for example, alkynes can be further activated by fluorination, substitution of aromatic rings, or introduction of heteroatoms into aromatic rings.

[0273] Figure 6 This paper depicts a specific example of payload site-specific coupling based on full-length IgG glycan remodeling and subsequent azide-cyclooctyne click chemistry. First, IgG is enzymatically remodeled by glycosylase-mediated pruning of all different glycoforms, followed by glycosyltransferase-mediated transfer of azide sugars to the core GlcNAc released by the glycosylase. In the next step, the azide-remodeled IgG is placed into an immune cell-conjugating peptide, which has been modified with a single cyclooctyne for metal-free click chemistry (SPAAC), resulting in a 2:2 bispecific antibody. The figure also depicts the cyclooctyne-peptide construct having a specific spacer between the cyclooctyne and the peptide, allowing for adjustment of the IgG-peptide distance or conferring other properties to the resulting bispecific antibody.

[0274] Figure 7 A two-stage approach, as described herein, is described in which a glycan-remodeled antibody with functional group F1 reacts with a bifunctional linker Q1-L-F2 (where click probes Q1 and F2 are non-reactive to each other) to perform a metal-free click chemistry reaction to form bond Z1. The bifunctional linker may contain F2 appearing 2 to 4 times (i.e., a trivalent, tetravalent, or pentavalent linker). In the second step, the antibody containing the reactive group F2 reacts with a click probe Q2, which is part of a linker-drug containing a drug appearing 1 to 3 times.

[0275] Figure 8 depicts three examples of bifunctional linkers Q1-L-F2 that satisfy the condition that Q1 and F2 are not reactive with each other, where Q1 is DBCO (as shown in A) or TMTHSI (as shown in B and C), and F2 is a tetrazine analog (as shown in AC).

[0276] Figure 9 Two examples of bifunctional linkers Q1-L-F2 are shown, satisfying the condition that Q1 and F2 are not reactive with each other, wherein Q1 is TMTHSI (as shown in D) or DBCO (as shown in E), the tetrazine is alkyl, methyltetrazine (as shown in D) or phenyltetrazine (as shown in E), and the linker is tetravalent (as shown in D) or pentavalent (as shown in E).

[0277] Figure 10An example of the bifunctional linker Q1-L-F2(F) is described, where Q1 is DBCO and F2 is an iminosydino ketone variant known to react very slowly with DBCO (k < 0.001 M). -1 s -1 ).

[0278] Figure 11 Three examples of the bifunctional linker Q1-L-F2 are shown, where F2 is a potential click-reactive group (i.e., not reactive itself, but needs to be chemically or enzymatically converted into a reactive click probe): for example, phenol or catechol in compound G (which can be converted into ortho-quinone after treatment with tyrosinase or NaIO4, respectively); serine in compound H (which can be converted into nitrone after treatment with NaIO4 and then with N-methylhydroxylamine); tetrazolium in compound I (which can be converted into nitrile imine after treatment with 200 nm light); or dihydrotetraazine in compound M (which can be converted into tetraazine after treatment with horseradish peroxidase or 660 nm light).

[0279] Figure 12 Several examples of the preferred connector-constructor Q1-L-F2 are shown.

[0280] Figure 13 , Figure 14 and Figure 15 The preferred connector-payload construct Q is shown. 2 (L B )D x Several examples, where x = 1 ( Figure 13 ), x = 2( Figure 14 ) and x = 3 ( Figure 15 ).

[0281] Figure 16 The relative retention times (rrt) of various ADCs relative to trastuzumab, measured by HIC-HPLC, were plotted, showing that rrt = 1.14 for DAR4, 1.20 for DAR8, and... The display shows rrt = 1.25.

[0282] Figure 17 The monomeric levels of various ADCs were depicted under physiological conditions (PBS, 37°C).

[0283] Figure 18A The complete RP-UPLC chromatogram of trastuzumab-(tetraazine)4 [trastuzumab-(45)2] is shown, and Figure 18B The changes in retention times of DAR species 2, 4, and 6 when coupled with 98 are shown.

[0284] Figure 19 shows the stability of DAR 8ADC in PBS at 37 °C over time (as a percentage compared to DAR at t=0). Figure 19A The results show that the DAR reduction is the same for all ADCs, regardless of the linker used. A reduction of <20% is very low for a 28-day timeframe, therefore the construct is stable. Figure 19B This shows that no clustering was observed during these 28 days.

[0285] Figure 20 shows the stability of the DAR 8 ADC in human serum over time at 37 °C. Similarly, all ADCs based on different linkers showed the same DAR reduction (A) (percentage compared to DAR at t=0) and aggregate formation (B).

[0286] Figure 21 shows the stability of the DAR8 ADC in mouse serum over time at 37 °C. Similarly, all ADCs based on different linkers showed the same DAR reduction (A) (percentage of DAR compared to t=0) and aggregate formation (B) (although showing large changes over time, the trend was the same).

[0287] Figure 22 shows in vitro potency data for NCI-N87(A), BT-474(B), and MDA-MB231(C) cells treated with ADCs trastuzumab-(3)2-(59)4 and trastuzumab-(3)2-(63)4 at progressively increasing concentrations and with the corresponding free payloads mitomycin C and gefitinib. Both DAR8 ADCs showed improved N87 cell killing compared to the corresponding free payloads, with IC50... 50 The values ​​are 0.2 nM and 0.5 nM, respectively, while the corresponding IC of the free effective payload is... 50 The values ​​were 94 nM and 997 nM. A similar trend was also observed in BT-474 cells compared to the corresponding free payload, where IC50 was 94 nM and 997 nM. 50 The values ​​are 0.2 nM and 0.5 nM, respectively, while the corresponding IC of the free effective payload is... 50 The values ​​were 905 nM and 410 nM. The negative control cell line MDA-MB-231 showed no efficacy and could not distinguish between the ADC and the free payload.

[0288] Figure 22 shows in vitro potency data for NCI-N87(A) and MDA-MB231(B) cells treated with increasing concentrations of DAR8 ADCs with different payloads. All ADCs showed similar cell-killing activity against NCI-N87 cells, with IC50 showing the highest efficiency. 50Values ​​ranged from 0.08 to 0.34 nM. The negative control cell line MDA-MB-231 showed no difference between the different ADCs and no cell killing.

[0289] Figure 24 shows an in vitro comparison of DAR among mitomycin ADCs. Figure 24A The changes in efficacy of higher DAR types compared to lower DAR types are shown. Figure 24B The negative control cell line MDA-MB-231 is shown, in which no significant differences were observed between ADCs.

[0290] Figure 25A In vivo efficacy data over time for the model expressing EMT6-hHER2 are presented. DAR4 showed a similar antitumor response compared to DAR8, but the dose level of the payload was adjusted. Figure 25B The weight of the mice over time is shown.

[0291] Figure 26A In vivo efficacy data over time for low-dose treatment (2 mg / kg) in tumor models expressing EMT6-hHER2 are presented. All DAR8 ADCs according to the invention showed similar antitumor responses, with improvements compared to the baseline Enhertu at the same dose level. Figure 26B The body weight of mice over time after low-dose treatment is shown. Figure 26C A graph showing high-dose treatment (6 mg / kg) with three ADCs is presented. All three tested DAR8 ADCs according to the invention showed complete remission up to day 30. Figure 26D The body weight of mice over time after high-dose treatment is shown. Figure 26E Survival rates of the test substance over time are shown in the Kaplan-Mayer plots for both low-dose and high-dose treatments. Significant effects were observed when the benchmark Enhertu was compared to the novel DAR8 ADC according to the invention.

[0292] Figure 27 In vivo tolerability data for female CD-1 mice over time are shown. At the highest concentration, only one mouse was euthanized due to severe weight loss (>20%). Therefore, the maximum tolerated dose is at least 80 mg / kg.

[0293] Figure 28 shows RP-UPLC chromatograms of the complete trastuzumab-(tetraazine)4[trastuzumab-(3)2](A) and the same construct coupled with 7(B) or 9(C). Figure 28B The chromatogram was generated before purification and contains some residual compound 3. Figure 28B and Figure 28CThe chromatogram showed that the DAR8 construct was almost completely formed, with only very small additional peaks. Example

[0294] The present invention is illustrated by the following examples. General reagents and analysis

[0295] General procedures for analytical RP-UPLC (for DTT-treated samples) Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL in PBS (pH 7.4)) was added to 12.5 mM DTT, 100 mM TrisHCl (pH 8.0) (40 μL) and incubated at 37 °C for 15 min. The reaction was quenched by adding 49% acetonitrile, 49% water, and 2% formic acid (50 μL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD column. The sample (5 μL) was injected at 0.4 mL / min onto a Bioresolve RP mAb 2.1*150 mm 2.7 μm (Waters) column at 70 °C. A linear gradient of 0.1% TFA and acetonitrile in water was applied from 30% to 54% over 9 min. The absorbance of the elution peak was measured at 215 nm, and the reaction conversion was determined by automatic integration (MassLynx, Waters Corporation).

[0296] A general procedure for mass spectrometry analysis of (modified) monoclonal antibodies: Prior to mass spectrometry analysis, IgG was treated with IdeS for Fc / 2 fragment analysis. To analyze both the light and heavy chains, 20 μg of modified IgG solution was incubated with 100 mM DTT at 37°C for 5 minutes, with a total volume of 4 μL. If present, azide functional groups were reduced to amines under these conditions. For Fc / 2 fragment analysis, 20 μg of modified IgG solution was incubated with IdeS / Fabricator. TM (1.25 U / μL) was incubated together in phosphate-buffered saline (PBS) (pH 6.6) at 37°C for 1 hour, with a total volume of 10 μL. The sample was diluted to 80 μL and then analyzed by electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF instrument. The deconvolution spectrum was obtained using Magtran software.

[0297] General procedures for analytical SEC analysis:HPLC-SEC analysis was performed on an Agilent 1100 series (Hewlett-Packard) Xbridge BEH200A column (3.5 μM, 7.8 x 300 mm, PN 186007640, Waters Corporation). Samples were diluted to 1 mg / mL in PBS and measured at 0.86 mL / min isocratic rate (100 mM sodium phosphate (NaHPO4 / Na2PO4), 200 mM NaCl, pH 6.8, containing 10% isopropanol) for 17 minutes.

[0298] A universal procedure for enzymatically remodeling IgG into mAb-(6-N3-GalNAc)2: The IgG (15 mg / mL) was mixed with 1% w / w EndoSH (as described in PCT / EP2017 / 052792, which is incorporated herein by reference, see Examples 1-3 and SEQ.ID No:1), 3% w / w His-TnGalNAcT (as described in PCT / EP2016 / 059194, which is incorporated herein by reference, see Examples 3 and 4 and SEQ.ID No:49), 0.01% AP (Roche) and UDP 6-N3-GalNAc ( Figure 3 Compound 2d (10 eq compared to IgG) was incubated in 6 mM MnCl2 and TBS at 30 °C for 16 h. Next, the functionalized IgG was purified using a HiTrap MabSelect 5 mL column. After loading the reaction mixture, the column was washed with TBS + 0.2% triton and TBS. IgG was eluted with 0.1 M AcOH (pH 2.7) and neutralized with 2.5 M Tris-HCl (pH 8.8). After three dialyses 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). Example 1-49: Synthesis of click probes and linker conjugates Example 1: Preparation of Compound 3

[0299] Compound 2 (10.3 mg, 3 Eq, 36.6 μmol) was added to a solution of compound 1 (10.0 mg, 1 Eq, 12.2 μmol) in dry DMF (850 μL), followed by the addition of triethylamine (6.18 mg, 8.51 μL, 5 Eq, 61.1 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by preparative HPLC (30% → 100%, MeCN / water + 10 mM NH4HCO3, run time 21 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 3 (5 mg, 4.7 μmol, 38%) as a pink solid. For C 57 H 67 N 12 O 10 + (M+H) + The calculated value of LCMS(ESI+) is 1079.5, and the measured value is 1079.9. Example 2: Preparation of Compound 5

[0300] Compound 4 (163 mg, 240 μmol) was added to a mixture of eczema 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 to 9 mL DCM and purified by silica gel rapid column chromatography (0→40% MeOH / DCM) to give compound 5 (155 mg, 159 μmol, 68%). For C 55 H 54 FN6O 10 + (M+H) + The calculated LCMS (ESI+) value was 977.39, and the measured value was 977.72. In addition to 5, the free base of eczema (82.4 mg, 189 μmol, 20%) was also recovered. For C... 24 H 23 FN3O4 + (M+H) + The calculated value of LCMS (ESI+) is 436.5, and the measured value is also 436.5. Example 3. Preparation of Compound 7

[0301] The synthesis of BCN-HS-PEG2-HS-(va-PABC-Ex)2(7) is also described in PCT / EP2021 / 075401 (Example 4) (which is incorporated herein by reference). A solution of Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 6 (65 mg, 72 μmol) in DMF (1.4 mL) were added to a solution of compound 5 (155 mg, 159 μmol) in DMF (1.6 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL), and purified by silica gel rapid column chromatography (0→40% MeOH / DCM) to give 7 (94 mg, 44 μmol, 28%) as a pale yellow solid. For C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + The calculated value of LCMS(ESI+) is 1066.9, and the measured value is 1067.1. Example 4. Preparation of Compound 9

[0302] The synthesis of BCN-HS-PEG2-(eva-PABC-Ex)2(9) is also described in PCT / EP2021 / 075401 (Example 11) (which is incorporated herein by reference). A solution of BCN-HS-PEG2-b-(Glu(OFm)-OH)2 (8, 12.1 mg, 10 μmol, 1.0 eq) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-ecisecan (5b, Fmoc deprotected 5, 19 mg, 25 μmol, 2.5 eq) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 eq), and HATU (8.9 mg, 23 μmol, 2.3 eq). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with DCM (800 μL) and purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give a product as a clear oil (the yield was difficult to determine due to the presence of DMF). For C 140 H 150 F2N 17 O 33 S +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 1334.0, and the measured value is 1334.8.

[0303] The compound was dissolved in DMF (300 μL), and triethylamine (21 μL, 150 μmol, 15 eq) was added. After 17 hours at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel rapid column chromatography (0% → 45% MeOH, in DCM) to give compound 9 as a yellow solid (10.2 mg, 4.4 μmol, 44%). For C 112 H 130 F2N 17 O 33 S +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 1156.2, and the measured value is 1156.7. Example 5. Preparation of Compound 11

[0304] Diethanolamine (894.1 mg, 816.5 μL, 1.1 Eq, 8.504 mmol) and sodium carbonate (1.188 g, 1.45 Eq, 11.21 mmol) were added to 1,4-dioxane (20 mL) and water (20 mL). The mixture was stirred at room temperature to form a suspension. (9H-fluorene-9-yl)methyl chloroformate (2000 mg, 1 Eq, 7.731 mmol) was dissolved in toluene (5 mL) and added dropwise to the above reaction solution (keeping the internal temperature below 25 °C during the addition), and stirring was continued for 2 hours. The pH was adjusted to 3 with 4 M HCl (aqueous), and the aqueous phase was extracted twice with ethyl acetate (50 mL). The organic phase was washed with saturated brine (2 x 50 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 10 (3.13 g, 7.6 mmol, 99%, 80% purity) as an opaque oil. For C 19 H 22 NO4 + (M+H) + The calculated value of LCMS(ESI+) is 328.2, and the measured value is 928.3.

[0305] Compound 10 (1260 mg, 1 Eq, 3.849 mmol) was dissolved in dry DCM (50 mL). Then, bis(perfluorophenyl) carbonate (3.185 g, 2.1 Eq, 8.082 mmol) was added, followed by DIPEA (1.492 g, 2.01 mL, 3 Eq, 11.55 mmol), and stirring was continued for 3 hours. The residue was then purified by silica gel rapid column chromatography (0→10% acetone, in DCM) to give compound 11 (2.5 g, 3.3 mmol, 87%, 100% purity) as a clear oil. For C33 H 20 F 10 NO8 + (M+H) + The calculated value of LCMS(ESI+) is 748.1, and the measured value is 748.4. Example 6. Preparation of Compound 13

[0306] A solution of 2,2'-(ethane-1,2-diylbis(oxy))bis(ethyl-1-amine) (3.40 g, 3.35 mL, 10 Eq, 22.9 mmol) in DCM (40 mL) was cooled to 0 °C. Di-tert-butyl dicarbonate (500 mg, 1 Eq, 2.29 mmol) was added, and the mixture was stirred at room temperature for 18 hours. The organic phase was washed with water (3 x 40 mL) until all unreacted 2,2'-(ethane-1,2-diylbis(oxy))bis(ethyl-1-amine) was extracted. The organic phase was dried (Na₂SO₄) and concentrated under vacuum to give 12 (361 mg, 1.3 mmol, 57%, 90% purity) as a yellow oil. For C 11 H 25 N2O4 + (M+H) + The calculated value of LCMS(ESI+) is 249.2, and the measured value is 249.4.

[0307] Compound 11 (109.6 mg, 2.2 Eq, 441.5 μmol) was added to a solution of amine 12 (150.0 mg, 1 Eq, 200.7 μmol) in dry DCM (5.00 mL), followed by the addition of DIPEA (129.7 mg, 175 μL, 5 Eq, 1.003 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum. The resulting crude product was dissolved in DCM (1 mL) and loaded onto a column for purification by silica gel rapid column chromatography (0→20% MeOH, in DCM) to give a colorless oily Boc-protected intermediate (218 mg, 0.20 mmol, 99%, 80% purity). For C 43 H 66 N5O 14 + (M+H) + The calculated value of LCMS(ESI+) is 876.5, and the measured value is 876.7.

[0308] HCl in dioxane (147 mg, 1.00 mL, 4 mol, 20 Eq, 4.02 mmol) was added to a solution of the intermediate (176 mg, 1 Eq, 201 μmol) in dry DCM (5.00 mL). The reaction mixture was stirred at room temperature for 18 hours and then concentrated under vacuum to give HCl salt 13 (150 mg, 200 μmol, 99%) as a grayish-white foam. For C 33 H 50 N5O 10 + (M+H) + The calculated value of LCMS (ESI+) is 676.4, and the measured value is 676.6. Example 7. Preparation of Compound 15

[0309] Di-tert-butyl dicarbonate (775 mg, 1.2 Eq, 3.55 mmol) was added to a solution of sulfonyl-D-alanine hydrate (554 mg, 1 Eq, 2.96 mmol) and triethylamine (599 mg, 825 μL, 2 Eq, 5.92 mmol) in DMF (15 mL). The reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under vacuum. The residue was redissolved in CH2Cl2 (10 mL) and treated with Et2O (30 mL) under vigorous stirring. The organic solvent was poured off and discarded. The oily residue was washed with Et2O (20 mL) and dried under vacuum to give compound 14 (1134 mg, 2.9 mmol, 100%, 70% purity) as a colorless oil. For C8H 14 NO7S - (MH) - The calculated value of LCMS(ESI-) is 268.1, and the measured value is 268.3.

[0310] Compound 13 (50 mg, 1 Eq, 67 μmol), HATU (76 mg, 3 Eq, 0.20 mmol), and DIPEA (52 mg, 70 μL, 6 Eq, 0.40 mmol) were added to a solution of N-Boc-sulfoalanine 14 (54 mg, 3 Eq, 0.20 mmol) in DMF (1 mL), and stirring was continued for 2 hours. The mixture was concentrated under vacuum, and the crude product was dissolved in DCM (10 mL). The mixture was purified by silica gel rapid column chromatography (0% → 70% MeOH, in DCM) to give a colorless oily Boc-protected intermediate (50 mg, 36 μmol, 53%, 84% purity). For C 49 H 75 N7O 22S2 + (M+H) + The calculated value of LCMS(ESI+) is 1178.4, and the measured value is 1178.7.

[0311] To a solution of the Boc-protected intermediate (50 mg, 1 Eq, 42 μmol) in dry DCM (2.00 mL) and MeOH (1.00 mL), HCl in 1,4-dioxane (46 mg, 0.32 mL, 4 mol, 30 Eq, 1.3 mmol) was added. The reaction mixture was stirred at room temperature for 26 hours, and then another HCl in 1,4-dioxane (46 mg, 0.32 mL, 4 mol, 30 Eq, 1.3 mmol) was added to the reaction mixture. Stirring was continued for another hour, and then the reaction mixture was concentrated under vacuum to give compound 15 (45 mg, 43 μmol, 100%) as a grayish-white solid. For C 39 H 60 N7O 18 S2 + (M+H) + The calculated value of LCMS(ESI+) is 978.3, and the measured value is 978.6. Example 8. Preparation of Compound 17

[0312] HATU (11 mg, 3 Eq, 29 μmol) and DIPEA (7.4 mg, 9.9 μL, 6 Eq, 57 μmol) were added to a solution of 4-(6-methyl-1,2,4,5-tetraazine-3-yl)butyric acid (4.3 mg, 2.5 Eq, 24 μmol) in DMF (1 mL). After 10 minutes, HCl salt 15 (10 mg, 1 Eq, 9.5 μmol) was added, and stirring was continued for 1 hour. The reaction mixture was concentrated under vacuum, and the crude product was dissolved in MeCN (1 mL). The solution was purified by reversed-phase chromatography (10% → 100%, MeCN in water) to give 16 (7 mg, 5 μmol, 50%, 95% purity) as a pink solid. For C 53 H 76 N 15 O 20 S2 + (M+H) + The calculated value of LCMS(ESI+) is 1306.5, and the measured value is 1306.7.

[0313] Triethylamine (11 mg, 15 μL, 20 Eq, 0.11 mmol) was added to a solution of compound 16 (7.0 mg, 1 Eq, 5.4 μmol) in DMF (0.200 mL) and H2O (10 μL). The reaction mixture was stirred for 18 hours until complete deprotection of Fmoc was observed. TMTHSI-OSu (2.7 mg, 1.5 Eq, 8.0 μmol) was added to the reaction mixture, and stirring was continued for 4 days. The reaction mixture was purified by preparative HPLC (30% → 100%, MeCN / water + 1% AcOH, run time 21 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 17 (2.5 mg, 1.9 μmol, 36%) as a pink solid. For C 49 H 78 N 16 O 20 S3 -2 (M / 2-H) - The calculated value of LCMS(ESI-) is 653.2, and the measured value is 653.6. Example 9. Preparation of Compound 19

[0314] To a solution of compound 11 (50.0 mg, 1 Eq, 66.9 μmol) in dry DMF (1.00 mL), 3-(2-(2-aminoethoxy)ethoxy)propionic acid (26.1 mg, 2.2 Eq, 147 μmol) was added, followed by the addition of DIPEA (43.2 mg, 334 μL, 5 Eq, 334 μmol). After stirring for 18 hours, the reaction mixture was concentrated under vacuum. The resulting crude product was dissolved in DCM (3 mL) and loaded onto a column for purification by silica gel rapid column chromatography (0–10% MeOH in DCM with 1% AcOH) to give 18 (55.3 mg, 75.4 μmol, 113%) as a colorless oil. For C 35 H 48 N3O 14 + (M+H) + The calculated value of LCMS(ESI+) is 734.3, and the measured value is 734.6.

[0315] DCC (32.3 mg, 2.3 Eq, 157 μmol) and 1-hydroxypyrrolidine-2,5-dione (18.0 mg, 2.3 Eq, 157 μmol) were added to a solution of compound 18 (50.0 mg, 1 Eq, 68.1 μmol) dissolved in DCM (2 mL) and DMF (2 mL). The reaction mixture was stirred at room temperature for 18 hours. Sulfo-L-alanine (34.6 mg, 3 Eq, 204 μmol) and DIPEA (105.6 mg, 142.4 μL, 12 Eq, 818 μmol) were added to the reaction mixture, and stirring was continued for 24 hours. The mixture was filtered, and the filtrate was concentrated under vacuum. The crude product was dissolved in water and MeCN and purified by reversed-phase chromatography (0→100% MeCN in H2O+1% AcOH) to give compound 19 (73.1 mg, 70.6 μmol, 104%) as a colorless oil. For C 41 H 56 N5O 22 S2 - (MH) - The calculated value of LCMS(ESI-) is 1034.3, and the measured value is 1034.7. Example 10. Preparation of Compound 21

[0316] DIPEA (7.49 mg, 10.1 μL, 6 Eq, 57.9 μmol) and HATU (9.18 mg, 2.5 Eq, 24.1 μmol) were added to compound 19 (10.0 mg, 1 Eq, 9.65 μmol) in DMF (0.5 mL). Subsequently, (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methylamine hydrochloride (5.05 mg, 2.2 Eq, 21.2 μmol) was added. The mixture was stirred for 1 hour, concentrated under vacuum, and the resulting crude product was dissolved in DCM (10 mL). The solution was purified by silica gel rapid column chromatography (0% → 30% MeOH, in DCM) to give compound 20 (2.4 mg, 1.7 μmol, 18%) as a pink solid. For C 61 H 74 N 15 O 20 S2 - (MH) - The calculated value of LCMS(ESI-) is 1400.5, and the measured value is 1400.7.

[0317] Triethylamine (1.7 mg, 2.4 μL, 10 Eq, 17 μmol) was added to compound 20 (2.4 mg, 1.0 Eq, 1.7 μmol) in DMF (0.1 mL). The reaction mixture was stirred for 42 hours until Fmoc deprotection was observed. TMTH-SI-OSu (0.70 mg, 1.2 Eq, 2.1 μmol) was added to the reaction mixture, and stirring was continued for 92 hours. LCMS analysis indicated the formation of 21. For C 57 H 79 N 16 O 20 S3 - (MH) - The calculated value of LCMS(ESI-) is 1403.5, and the measured value is 1403.6. Example 11. Preparation of compound 23

[0318] HATU (61 mg, 3 Eq, 0.16 mmol) and DIPEA (55 mg, 74 μL, 8 Eq, 0.43 mmol) were added to a solution of 4-(6-methyl-1,2,4,5-tetraazine-3-yl)butyric acid (24 mg, 2.5 Eq, 0.13 mmol) in DMF (1 mL). After 10 minutes, compound 13 (40 mg, 1 Eq, 53 μmol) was added, and stirring was continued for 1 hour. The reaction mixture was purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM) to yield a pink solid Fmoc-protected intermediate (82 mg, 53 μmol, 97%, 65% purity). For C 47 H 66 N 13 O 12 + (M+H) + The calculated value of LCMS(ESI+) is 1004.5, and the measured value is 1004.8.

[0319] Piperidine (0.18 g, 0.21 mL, 40 Eq, 2.1 mmol) was added to a solution of the Fmoc-protected intermediate (82 mg, 65% wt, 1 Eq, 53 μmol) in DCM (1.0 mL), and stirring was continued for 22 hours. The reaction mixture was extracted with saturated NaHCO3, dried, and concentrated under vacuum to give 42 mg of a pink crude product. The crude product was dissolved in DCM (1 mL) and purified by silica gel rapid column chromatography (0% → 15% MeOH, in DCM) to give compound 22 (11.7 mg, 15.0 μmol, 28%) as a pink solid. For C 32 H56 N 13 O 10 + (M+H) + The calculated value of LCMS(ESI+) is 782.4, and the measured value is 782.7.

[0320] DBCO-C6-OSu (4.8 mg, 1.5 Eq, 11 μmol), HOBt (1.7 mg, 1.5 Eq, 11 μmol), and triethylamine (3.8 mg, 5.2 μL, 5 Eq, 37 μmol) were added to a solution of compound 22 (5.8 mg, 1 Eq, 7.4 μmol) in DMF (0.50 mL). The resulting reaction mixture was allowed to stand for 3 days, then diluted to 550 μL DMF and purified by preparative HPLC (injection volume 600 μL DMF; 5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 21 min, Xbridge prep C185 μm OBD column, 30 x 100 mm) to give compound 23 (1.6 mg, 1.5 μmol, 20%) as a pink oil. For C 53 H 73 N 14 O 12 + (M+H) + The calculated value of LCMS(ESI+) is 1097.5, and the measured value is 1097.8. Example 12. Preparation of Compound 25

[0321] 2-(2-aminoethoxy)ethanol-1-ol (185.9 mg, 177.4 μL, 3 Eq, 1.768 mmol) was added to a solution of TMTHSI-OSu (200.6 mg, 1 Eq, 589.3 μmol) in DCM (3 mL). The reaction mixture was stirred for 21 hours and subsequently purified by silica gel rapid column chromatography (0% → 60% Aceton, in DCM) to yield unactivated alcohol (168.2 mg, 509.0 μmol, 86.37%) as a colorless oil. For C 15 H 27 N2O4S + (M+H) + The calculated value of LCMS(ESI+) is 331.2, and the measured value is 331.1.

[0322] Unactivated alcohol (168.2 mg, 1.0 Eq, 509.0 μmol) was dissolved in DCM (3.00 mL), followed by the addition of bis(4-nitrobenzene) carbonate (309.7 mg, 2 Eq, 1.018 mmol) and triethylamine (154.5 mg, 213 μL, 3.0 Eq, 1.527 mmol). The solution was stirred for 2 hours and subsequently purified by silica gel rapid column chromatography (0% → 30% Aceton, in DCM) to yield compound 24 (262.5 mg, 0.41 mmol, 80%, 77% purity). For C 22 H 30 N3O8S + (M+H) + The calculated value of LCMS(ESI+) is 496.2, and the measured value is also 496.2.

[0323] Compound 24 (5.5 mg, 1.5 Eq, 11 μmol), HOBt (1.1 mg, 1 Eq, 7.4 μmol), and triethylamine (3.8 mg, 5.2 μL, 5 Eq, 37 μmol) were added to a solution of compound 22 (5.8 mg, 1 Eq, 7.4 μmol) in DMF (0.50 mL). The resulting reaction mixture was stirred for 18 hours and subsequently purified by preparative HPLC (injection volume 600 μL DMF; 30% → 100%, MeCN / water + 10 mM NH4HCO3, run time 21 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 25 (3.9 mg, 3.4 μmol, 46%) as a pink oil. For C 48 H 80 N 15 O 15 S + (M+H) + The calculated value of LCMS(ESI+) is 1138.6, and the measured value is 1138.8. Example 13. Preparation of Compound 26

[0324] To a solution of compound 1 (9.35 mg, 1 Eq. 11.4 μmol) in anhydrous DMF (500 μL), an HCl salt of methyltetraazine-PEG4-amine (9.8 mg, 2.1 Eq. 25 μmol) was added, followed by the addition of triethylamine (5.2 mg, 7.2 μL, 4.5 Eq. 52 μmol). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with 200 μL of DMF and purified by preparative HPLC (30% → 70%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 26 (9.1 mg, 6.9 μmol. 61%) as a pink oil. For C 67 H 87 N 12 O 16 + (M+H) + The calculated value of LCMS (ESI+) is 1315.6, and the measured value is 1315.8. Example 14. Preparation of Compound 27

[0325] To a solution of compound 1 (8.5 mg, 1.0 Eq, 10 μmol) in dry DMF (500 μL), an HCl salt of methyltetraazine-benzylamine (7.4 mg, 3.0 Eq, 31 μmol) was added, followed by the addition of triethylamine (5.3 mg, 7.2 μL, 5.0 Eq, 52 μmol). The reaction mixture was stirred at room temperature for 75 min, followed by purification by preparative HPLC (30% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 27 (6.1 mg, 5.9 μmol, 57%, 97% purity) as a pink solid. For C 53 H 59 N 12 O8 + (M+H) + The calculated value of LCMS (ESI+) is 991.5, and the measured value is 991.7. Example 15. Preparation of Compound 29

[0326] Diethanolamine (8.91 mg, 1.06 Eq, 84.7 μmol) and triethylamine (49 mg, 67 μL, 6.0 Eq, 0.48 mmol) were added to a solution of compound 24 (49.4 mg, 80% Wt, 1 Eq, 79.7 μmol) in anhydrous DCM (500 μL). After stirring at room temperature for 21 hours, bis(4-nitrobenzene) carbonate (65.4 mg, 2.70 Eq, 215 μmol) was added to the reaction mixture. After stirring for another 2 hours, the reaction mixture was purified by silica gel rapid column chromatography (0% → 20% Aceton, in DCM) to give compound 28 (40.9 mg, 35 μmol, 43%, 67% purity) as a yellow oil. For C 34 H 42 N5O 15 S + (M+H) + The calculated value of LCMS (ESI+) is 792.2, and the measured value is 792.5.

[0327] Compound 28 (7.9 mg, 67% Wt, 1 Eq, 6.7 μmol) was dissolved in anhydrous DMF, followed by the addition of compound 2 (4.5 mg, 2.4 Eq, 16 μmol) and triethylamine (3.4 mg, 4.7 μL, 5 Eq, 33 μmol). After stirring at room temperature for 24 hours, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 29 (5.3 mg, 5.3 μmol, 79%) as a pink oil. For C 46 H 62 N 13 O 11 + (M+H) + The calculated value of LCMS(ESI+) is 1004.4, and the measured value is 1004.6. Example 16. Preparation of Compound 31

[0328] A solution of Boc-Glu(OFm)-OH (639 mg, 1 Eq, 1.50 mmol) in DCM (5 mL) was cooled to 0 °C, followed by the addition of trifluoroacetic acid (3.08 g, 2.08 mL, 18 Eq, 27.0 mmol). The reaction mixture was stirred for 19 hours and then concentrated under vacuum and co-evaporated with DCM (6 x 5 mL) to give a TFA salt of NH-Glu(Fm)-OH as a grayish-white solid (569.9 mg, 1.30 mmol, 86.6%). For C 19 H 20 NO4 + (M+H) + The calculated value of LCMS(ESI+) is 326.2, and the measured value is 326.3.

[0329] To a solution of compound 28 (10.0 mg, 1 Eq, 13 μmol) in anhydrous DMF (250 μL) and anhydrous DCM (250 μL), a TFA salt of NH-Glu(Fm)-OH (14 mg, 2.5 Eq, 32 μmol) and DIPEA (9.8 mg, 13 μL, 6 Eq, 76 μmol) were added. After stirring at room temperature for 15 hours, the reaction mixture was purified by silica gel rapid column chromatography (0% → 30% MeOH, in DCM) to give compound 30 (14.2 mg, 12 μmol, 95%, 98% purity). For C 60 H 70 N5O 17 S + (M+H) + The calculated value of LCMS (ESI+) is 1164.5, and the measured value is 1164.7.

[0330] HATU (5.1 mg, 2.1 Eq, 13 μmol) and DIPEA (4.9 mg, 6.6 μL, 6 Eq, 38 μmol) were added to a solution of compound 30 (7.3 mg, 1 Eq, 6.3 μmol) in DCM (500 μL). After stirring for 5 minutes at room temperature, the HCl salt of methyltetraazine-benzylamine (3.4 mg, 2.3 Eq, 14 μmol) and anhydrous DMF (200 μL) were added to the reaction mixture. After stirring for 3 hours at room temperature, the reaction mixture was purified by silica gel rapid column chromatography (0→70% EtOAc, in heptane, followed by 0%→10% MeOH, in DCM) to give the Fmoc-protected tetraazine intermediate (3.6 mg, 1.5 μmol, 23%, 62% purity). For C 80 H 88 N 15 O 15 S +(M+H) + The calculated value of LCMS(ESI+) is 1530.6, and the measured value is 1530.8.

[0331] A Fmoc-protected tetrazine intermediate (3.6 mg, 1.5 μmol, 23%, 62% purity) was dissolved in anhydrous DMF (300 μL), followed by the addition of piperidine (8.0 mg, 40 Eq, 94 μmol). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with DMF (200 μL) and purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 31 (0.6 mg, 0.5 μmol, 20%, 95% purity) as a pink oil. For C 52 H 68 N 15 O 15 S + (M+H) + The calculated value of LCMS (ESI+) is 1174.5, and the measured value is 1174.6. Example 17. Preparation of compound 33

[0332] HATU (22 mg, 3.4 Eq, 58 μmol) was added to a solution of compound 14 (11.4 mg, 1.7 Eq, 29 μmol) in dry DMF (200 μL), followed by DIPEA (17 mg, 24 μL, 8.0 Eq, 134 μmol). After 30 minutes, the HCl salt of methyltetraazine-benzylamine (4.0 mg, 1.0 Eq, 17 μmol) was added. The reaction mixture was stirred at room temperature for 5 hours, concentrated under vacuum, and purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give a Boc-protected compound (4.5 mg, 9.9 μmol, 59%, 97% purity) as a pink solid. For C 18 H 23 N6O6S - (MH) - The calculated value of LCMS(ESI-) is 451.1, and the measured value is also 451.1.

[0333] The Boc-protected compound (2.2 mg, 97% wt, 1.0 Eq, 4.9 μmol) was dissolved in DCM (200 μL), and TFA (22 mg, 15 μL, 40 Eq, 190 μmol) was added. The reaction mixture was stirred at room temperature for 1 hour and concentrated under vacuum to give compound 32 (2.3 mg, 4.9 μmol, 100%, 100% purity) as a pink oil. Assuming the yield is quantifiable, compound 32 can be used in the next step without further purification. For C 13 H 15 N6O4S - (MH) - The calculated value of LCMS(ESI-) is 351.1, and the measured value is 351.0.

[0334] Compound 32 (3.8 mg, 1.8 Eq, 8.1 μmol) was added to a solution of compound 28 (3.5 mg, 1.0 Eq, 4.4 μmol) in dry DMF (300 μL), followed by the addition of Et3N (8.8 mg, 12 μL, 20 Eq, 20 μmol) and HOBt (0.7 mg, 1.0 Eq, 4.4 μmol). The reaction mixture was stirred at room temperature for 20 h and then purified by preparative HPLC (30% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 33 (0.5 mg, 0.4 μmol, 9%, 100% purity) as a pink solid. For C 48 H 62 N 15 O 17 S3 - (MH) - The calculated value of LCMS(ESI-) is 1216.4, and the measured value is 1216.7. Example 18. Preparation of compound 35

[0335] N-Boc-ethylenediamine (10.3 mg, 10.2 μL, 6.85 Eq, 64.4 μmol) was added to a solution of compound 28 (10.2 mg, 73% Wt, 1 Eq, 9.40 μmol) in anhydrous DCM (500 μL). After stirring at room temperature for 1 hour, the reaction mixture was purified by silica gel rapid column chromatography (0% → 30% EtOAc, in DCM, followed by 0% → 10% MeOH, in DCM) to give the Boc-protected intermediate (7.7 mg, 8.7 μmol, 92%, 93.7% purity). For C36 H 64 N7O 13 S + (M+H) + The calculated value of LCMS(ESI+) is 834.4, and the measured value is 834.3.

[0336] The Boc-protected intermediate (7.2 mg, 1 Eq, 8.6 μmol) was dissolved in anhydrous DCM (300 μL), followed by the addition of TFA (20 mg, 13 μL, 20 Eq, 0.17 mmol). The mixture was stirred at room temperature for 1 hour and then concentrated under vacuum and co-evaporated with toluene to give compound 34 (7.2 mg, 8.7 μmol, 100%). Assuming the yield was quantifiable, the product was used for the next step without any further purification. For C 26 H 48 N7O9S + (M+H) + The calculated value of LCMS(ESI+) is 634.3, and the measured value is also 634.3.

[0337] HATU (4.4 mg, 2.7 Eq, 12 μmol) and DIPEA (3.4 mg, 4.5 μL, 6 Eq, 26 μmol) were added to a solution of 4-(6-methyl-1,2,4,5-tetraazine-3-yl)butyric acid (2.0 mg, 2.5 Eq, 11 μmol) in anhydrous DMF (150 μL). After stirring at room temperature for 30 min, a solution of compound 34 (3.6 mg, 1 Eq, 4.3 μmol) in anhydrous DMF (150 μL) was added. After another 45 min, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 35 (2.2 mg, 1.7 μmol, 39%, 74% purity) as a pink oil. For C 40 H 64 N 15 O 11 S + (M+H) + The calculated value of LCMS (ESI+) is 962.5, and the measured value is 962.7. Example 19. Preparation of Compound 37

[0338] HCl salt of EDC (77.1 mg, 2.1 Eq, 338 μmol) and HOBt (49.3 mg, 2 Eq, 322 μmol) were added to a solution of Fmoc-Ser-OH (52.7 mg, 1 Eq, 161 μmol) in anhydrous DMF (1 mL). After stirring for 10 min at room temperature, N-Boc-ethylenediamine (4.0 mg, 3.9 μL, 1.2 Eq, 25 μmol) was added. The reaction mixture was stirred for 1 h at room temperature and subsequently purified by silica gel rapid column chromatography (0% → 5% MeOH, in DCM). The residue was ground with DCM to give a Boc-protected intermediate as a white solid (53.7 mg, 85.2 μmol, 52.9%, 74.5% purity). For C 25 H 32 N3O6 + (M+H) + The calculated value of LCMS(ESI+) is 470.2, and the measured value is 470.5.

[0339] The Boc-protected intermediate (53.7 mg, 74.5% Wt, 1 Eq, 85.2 μmol) was dissolved in anhydrous DCM (2 mL), followed by the addition of trifluoroacetic acid (194 mg, 131 μL, 20 Eq, 1.70 mmol). The mixture was stirred at room temperature for 2.5 h and then concentrated under vacuum and co-evaporated with toluene to give compound 36 (39.8 mg, 85.1 μmol, 100%). Assuming the yield was quantifiable, the product was used for the next step without any further purification. For C 20 H 24 N3O4 + (M+H) + The calculated value of LCMS(ESI+) is 370.2, and the measured value is also 370.2.

[0340] Compound 1 (12.8 mg, 1.9 Eq, 15.7 μmol) and DIPEA (5.2 mg, 7.1 μL, 5 Eq, 41 μmol) were added to a solution of compound 36 (9.5 mg, 2.5 Eq, 20 μmol) in anhydrous DCM (580 μL). After stirring at room temperature for 75 min, complete conversion to the Fmoc-protected DBCO intermediate was observed, after which piperidine (14 mg, 16 μL, 20 Eq, 0.16 mmol) was added to the reaction mixture. After stirring for 4 hours, the reaction mixture was concentrated under vacuum, and the residue was partitioned between water and DCM. The aqueous layer was evaporated under vacuum, and the crude product was transferred to an Eppendorf tube. The residue was dissolved in water (500 μL) and DMF (400 μL), the resulting solution was centrifuged, and the supernatant was purified by preparative HPLC (5% → 50%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 37 (1.4 mg, 1.4 μmol, 17%, 86% purity). For C 43 H 61 N8O 12 - (MH) - The calculated value of LCMS(ESI-) is 881.4, and the measured value is 881.3. Example 20. Preparation of compound 38

[0341] A solution of compound 36 (22.5 mg, 3.75 Eq, 47.6 μmol) in anhydrous DCM (750 μL) was added to a solution of compound 28 (17 mg, 61% Wt, 1 Eq, 13 μmol) in anhydrous DCM (500 μL), followed by the addition of DIPEA (14.7 mg, 20 μL, 8.75 Eq, 0.11 mmol). After stirring at room temperature for 26 hours, the solution was concentrated under vacuum, and the residue was dissolved in DMF (600 μL) and purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give the Fmoc-protected TMTHSI intermediate (8.5 mg, 6.8 μmol, 53%). For C 62 H 78 N9O 17 S + (M+H) +The calculated value of LCMS(ESI+) is 1252.5, and the measured value is 1252.8.

[0342] The Fmoc-protected TMTHSI intermediate (8.5 mg, 1 eq, 6.8 μmol) was dissolved in anhydrous DMF (450 μL), followed by the addition of piperidine (23 mg, 27 μL, 40 Eq, 0.27 mmol). After stirring for 2 hours, the reaction mixture was purified by reversed-phase chromatography (10% → 100%, MeCN / water + 10 mM NH4HCO3) to give compound 38 (1.8 mg, 2.2 μmol, 33%). For C 32 H 58 N9O 13 S + (M+H) + The calculated value of LCMS(ESI+) is 808.4, and the measured value is 808.7. Example 21. Preparation of compound 41

[0343] CSI (260 mg, 159 μL, 1.1 Eq, 1.83 mmol) was added to a solution of (R)-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropane-2-yl)carbamate tert-butyl ester (509 mg, 1.0 Eq, 1.67 mmol) in dry DCM (10 mL), and the reaction mixture was stirred at room temperature for 10 min. Et3N (506 mg, 697 μL, 3.0 Eq, 5.00 mmol) was added, followed by m-PEG4-NH2 (380 mg, 1.1 Eq, 1.83 mmol). The reaction mixture was stirred at room temperature for 3 h and subsequently purified by silica gel rapid column chromatography (0% → 25% MeOH, in DCM) to give a clear oily compound with Boc and TBS protection (1.01 g, 1.5 mmol, 90%, 92% purity). For C 19 H 44 N3O 11 SSi + (M+H-Boc) + The calculated value of LCMS(ESI+) is 518.3, and the measured value is 518.5.

[0344] Mix MeOH (8.7g, 11mL, 5.10) 2The mixture (Eq, 273 mmol) was cooled on ice, followed by the dropwise addition of acetyl chloride (1.28 g, 1.16 mL, 30 Eq, 16.3 mmol). The reaction mixture was stirred at 0 °C for 5 minutes. The ice bath was removed, and a solution of the Boc and TBS-protected compound (337 mg, 92% Wt, 1.0 Eq, 545 μmol) in dry DCM (1.5 mL) was added. The reaction mixture was stirred at room temperature for 18 hours and concentrated under vacuum to give compound 39 (196 mg, 446 μmol, 82%, 100% purity). For C 13 H 30 N3O9S + (M+H) + The calculated value of LCMS (ESI+) is 404.1, and the measured value is 404.4.

[0345] Compound 39 (177 mg, 5.0 Eq, 402 μmol) was added to a solution of compound 11 (60 mg, 1.0 Eq, 80 μmol) in dry DCM (3000 μL), followed by the addition of DIPEA (62 mg, 84 μL, 6.0 Eq, 482 μmol). The reaction mixture was stirred at room temperature for 22 hours and subsequently purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM) to give the diol compound (72 mg, 53 μmol, 66%, 87% purity). For C 47 H 76 N7O 24 S2 + (M+H) + The calculated value of LCMS(ESI+) is 1186.4, and the measured value is 1186.7.

[0346] A solution of a diol compound (5.0 mg, 87% wt, 1.0 Eq, 3.7 μmol) in dry DCM (200 μL) was supplemented with bis-PNP carbonate (3.3 mg, 3.0 Eq, 11 μmol), followed by the addition of DIPEA (2.8 mg, 3.8 μL, 6.0 Eq, 22 μmol). The reaction mixture was stirred at room temperature for 19 hours. An HCl salt of methyltetraazine-benzylamine (2.4 mg, 2.8 Eq, 10 μmol) was then added. The reaction mixture was stirred at room temperature for 3 hours and subsequently purified by silica gel rapid column chromatography (0% → 15% MeOH, in DCM) to give compound 40 (2.6 mg, 1.6 μmol, 43%, 100% purity). For C 69 H 97 N 18 O 26 S2 + (M+NH4)+ The calculated value of LCMS (ESI+) is 1657.6, and the measured value is 1658.0.

[0347] To a solution of compound 40 (2.6 mg, 1.0 Eq, 1.6 μmol) in DCM (200 μL), piperidine (5.4 mg, 6.3 μL, 40 Eq, 63 μmol) was added. The reaction mixture was stirred at room temperature for 4 hours. It was purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give the Fmoc-deprotected compound (2.5 mg, 1.6 μmol, 100%, 92% purity). For C 54 H 97 N 17 O 24 S2 + (M+H) + The calculated value of LCMS(ESI+) is 1418.5, and the measured value is 1419.0.

[0348] Compound 24 (1.3 mg, 1.5 Eq, 2.6 μmol) was added to a solution of Fmoc-deprotected compound (2.5 mg, 1.0 Eq, 1.8 μmol) in DMF (200 μL), followed by the addition of Et3N (1.8 mg, 2.4 μL, 10 Eq, 18 μmol) and HOBt (0.4 mg, 1.5 Eq, 2.6 μmol). The reaction mixture was stirred at room temperature for 23 hours and then purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 41 (0.6 mg, 0.3 μmol, 20%, 100% purity) as a pink solid. For C 70 H 109 N 19 O 29 S3 +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 887.8, and the measured value is 887.9. Example 22. Preparation of compound 45

[0349] HCl salt of EDC (228.7 mg, 1.2 Eq, 1.002 mmol) and HOAt (34.10 mg, 250.5 μL, 1 mol, 0.3 Eq, 250.5 μmol) were added to a solution of Fmoc-NH-PEG2-propionic acid (400.3 mg, 1.2 Eq, 1.002 mmol) in anhydrous DCM (5 mL). After stirring at room temperature for 30 minutes, a solution of tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (400.3 mg, 1.2 Eq, 1.002 mmol) in anhydrous DMF (1 mL) was added. The reaction mixture was stirred at room temperature for 75 minutes, then the DCM was evaporated, and the residue was purified by reversed-phase chromatography (10% → 100%, MeCN / water + 10 mM NH4HCO3) to give compound 42 (585.4 mg, 628 μmol, 75.2%, 95.2% purity) as a colorless oil. For C 47 H 71 N2O 14 + (M+H) + The calculated value of LCMS(ESI+) is 887.5, and the measured value is 887.8.

[0350] Compound 42 (585.4 mg, 95% Wt, 1 Eq, 626.9 μmol) was dissolved in anhydrous DCM (1.5 mL), followed by the addition of trifluoroacetic acid (2.2 g, 1.5 mL, 31 Eq, 19 mmol). After stirring at room temperature for 2 hours, DCM (10 mL) was added, and the reaction mixture was extracted with 1 M HCl (3 x 10 mL). The combined aqueous layers were extracted with DCM (2 x 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give compound 43 (327.7 mg, 0.41 mmol, 65%, 90% purity) as a pale yellow gel. For C 35 H 47 N2O 14 + (M+H) + The calculated value of LCMS(ESI+) is 719.3, and the measured value is 719.7.

[0351] The HCl salt of EDC (24.8 mg, 4.09 Eq, 109 μmol) and HOBt (16.7 mg, 4.11 Eq, 109 μmol) were added to a solution of compound 43 (21.2 mg, 90% wt, 1.0 Eq, 26.5 μmol) in anhydrous DCM (400 μL). After stirring at room temperature for 90 min, the HCl salt of methyltetraazine-benzylamine (25.2 mg, 3.99 Eq, 106 μmol) was added. The reaction mixture was stirred for 15 min and subsequently purified by silica gel rapid column chromatography (0% → 15% MeOH, in DCM) to give a pink oily Fmoc-protected tetraazine intermediate (15.5 mg, 11 μmol, 43%, 94% purity). For C 65 H 74 N 17 O 11 + (M+H) + The calculated value of LCMS(ESI+) is 1268.6, and the measured value is 1269.1.

[0352] The Fmoc-protected tetrazine intermediate (15.5 mg, 95.0% wt, 1 Eq, 11.6 μmol) was dissolved in anhydrous DCM (200 μL), followed by the addition of piperidine (43 mg, 50 μL, 44 Eq, 0.51 mmol). After stirring at room temperature for 90 min, the reaction mixture was purified by silica gel rapid column chromatography (0% → 35% MeOH, in DCM) to give compound 44 (11.8 mg, 9.9 μmol, 86%, 88% purity) as a pink solid. For C 50 H 64 N 17 O9 + (M+H) + The calculated value of LCMS (ESI+) is 1046.5, and the measured value is 1046.9.

[0353] DBCO-NHS ester (8.0 mg, 1.5 Eq, 20 μmol) was added to a solution of compound 44 (15 mg, 92% Wt, 1 Eq, 13 μmol) in anhydrous DMF (450 μL), followed by the addition of DIPEA (8.5 mg, 11 μL, 5 Eq, 66 μmol). After stirring at room temperature for 1 hour, the reaction mixture was further diluted with 250 μL of DMF and purified by preparative HPLC (30% → 95%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm). The resulting residue was dissolved in DCM (1 mL) and precipitated by the addition of diethyl ether (15 mL). The precipitate was washed with diethyl ether (3 x 5 mL) and concentrated under vacuum to give compound 45 (9.5 mg, 7.1 μmol, 54%) as a pink solid. For C 69 H 77 N 18 O 11 + (M+H) + The calculated value of LCMS(ESI+) is 1333.6, and the measured value is 1333.7. Example 23. Preparation of compound 46

[0354] To a solution of compound 44 (3.0 mg, 1.0 Eq, 2.7 μmol) in dry DMF (200 μL), TMTHSI-OSu (3.3 mg, 3.6 Eq, 9.6 μmol) was added, followed by the addition of triethylamine (2.6 mg, 3.8 μL, 10 Eq, 26 μmol) and HOBt (0.5 mg, 1.2 Eq, 3.2 μmol). The reaction mixture was stirred at room temperature for 26 h and subsequently purified by preparative HPLC (30% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 46 (0.6 mg, 0.4 μmol, 20%, 93% purity) as a pink solid. For C 61 H 79 N 18 O 11 S + (M+H) + The calculated value of LCMS(ESI+) is 1271.6, and the measured value is 1271.8. Example 24. Preparation of compound 49

[0355] To a solution of compound 47 (25.4 mg, 1 Eq, 24.0 μmol, commercially available from GenScript Biotech) in anhydrous DMF (500 μL), methyltetraazine-NHS ester (20.3 mg, 2.59 Eq, 62.0 μmol) was added, followed by DIPEA (21.5 mg, 29.0 μL, 6.95 Eq, 166 μmol). After stirring for 30 minutes, the desired product was precipitated by adding diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 x 1.5 mL) and concentrated under vacuum to give an alloc-protected tetraazine intermediate (57.0 mg, 21.9 μmol, 91.2%, 49.4% purity) as a pink solid. For C 63 H 70 N 17 O 14 + (M+H) + The calculated value of LCMS (ESI+) is 1288.5, and the measured value is 1288.9.

[0356] Alloc-protected tetrazine intermediate (22.9 mg, 49.4% Wt, 1 Eq, 8.78 μmol) was dissolved in anhydrous DMF (500 μL), followed by the addition of tetra(triphenylphosphine)palladium(0) (1.26 mg, 179 μL, 6.1 mmol, 0.124 Eq, 1.09 μmol) and pyrrolidine (2.0 mg, 2.3 μL, 3.2 Eq, 28 μmol). After 10 minutes, the reaction mixture was purified by reversed-phase chromatography (10% → 100%, MeCN / water + 10 mM NH4HCO3) to give compound 48 as a pink solid (13.2 mg, 5.9 μmol, 67%, 54% purity). For C 57 H 61 N 17 O 14 + (M+H) + The calculated value of LCMS(ESI+) is 1208.5, and the measured value is 1209.0.

[0357] Compound 48 (13.2 mg, 54% Wt, 1 Eq, 5.90 μmol) was dissolved in anhydrous DMF (350 μL), followed by the addition of piperidine (10.0 mg, 11.7 μL, 20 Eq, 118 μmol). After 35 minutes, the reaction mixture was purified by reversed-phase chromatography (10% → 100%, MeCN / water + 10 mM NH4HCO3) to give a pink solid of the deprotected Fmoc intermediate (3.4 mg, 3.3 μmol, 56%). For C 42 H 52 N 17 O 12 + (M+H) + The calculated value of LCMS (ESI+) is 986.4, and the measured value is 986.7.

[0358] The deprotected intermediate of Fmoc (3.4 mg, 1 Eq, 3.3 μmol) was added to a solution of anhydrous DMF (200 μL) with DBCO-NHS ester (3.1 mg, 2.3 Eq, 7.7 μmol) and DIPEA (2.1 mg, 2.9 μL, 5 Eq, 17 μmol). After 1 hour, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridgeprep C18 5 μm OBD column, 30 x 100 mm) to give compound 49 (0.7 mg, 0.5 μmol, 20%, 94% purity) as a pink oil. For C 61 H 65 N 18 O 14 + (M+H) + The calculated value of LCMS(ESI+) is 1273.5, and the measured value is 1273.8. Example 25. Preparation of Compound 51

[0359] A amino-PEG8-OH (6.5 mg, 3 Eq, 18 μmol) was added to a solution of compound 48 (7.1 mg, 1 Eq, 5.9 μmol) in anhydrous DMF (300 μL), followed by the addition of DIPEA (4.6 mg, 6.1 μL, 6 Eq, 35 μmol) and HATU (4.9 mg, 2.2 Eq, 13 μmol). After 45 minutes, the desired product was precipitated by adding diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 x 1.5 mL) and concentrated under vacuum to give compound 50 (31.4 mg, 10 μmol, 170%, 62% purity) as a pink solid. Assuming the yield was quantifiable, the product was used for the next step without any further purification. For C 89 H 126 N 19 O 28 - (MH) - The calculated value of LCMS(ESI-) is 1908.9, and the measured value is 1908.6.

[0360] Compound 50 (11 mg, 1 Eq, 5.8 μmol) was dissolved in anhydrous DMF (300 μL), followed by the addition of piperidine (9.8 mg, 11 μL, 20 Eq, 0.12 mmol). After 2.5 hours, the desired product was precipitated by adding diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 x 1.5 mL) and concentrated under vacuum to give a pink solid of the deprotected Fmoc intermediate (8.0 mg, 3.2 μmol, 55%, 67% purity). For C 74 H 116 N 19 O 26 - (MH) - The calculated value of LCMS(ESI-) is 1686.8, and the measured value is 1687.2.

[0361] The deprotected intermediate of Fmoc (8.0 mg, 1 Eq, 4.7 μmol) was added to a solution of anhydrous DMF (350 μL) with DBCO-NHS ester (2.9 mg, 1.5 Eq, 7.1 μmol) and DIPEA (3.1 mg, 4.1 μL, 5 Eq, 24 μmol). After 1 hour, the reaction mixture was purified by preparative HPLC (30% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridgeprep C18 5 μm OBD column, 30 x 100 mm). The residue was dissolved in DMF (500 μL) and precipitated by adding diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 x 1.5 mL) and concentrated under vacuum to give compound 51 (2.8 mg, 1.3 μmol, 27%, 90% purity) as a purple solid. For C 93 H 132 N 20 O 28 +2 (M / 2+H) + The calculated value of LCMS (ESI+) is 988.5, and the measured value is 988.7. Example 26. Preparation of Compound 54

[0362] To a solution of compound 52 (25.0 mg, 1 Eq, 17.0 μmol, commercially available from Genscript Biotech) in anhydrous DMF (500 μL), methyltetraazine-NHS ester (19.6 mg, 3.52 Eq, 59.9 μmol) was added, followed by DIPEA (15.4 mg, 20.7 μL, 7 Eq, 119 μmol). After stirring for 2 hours, the reaction mixture was diluted with DMF (4 mL), and the desired product was precipitated by adding diethyl ether (10 mL). The precipitate was washed with diethyl ether (3 x 5 mL) and concentrated under vacuum to give an alloc-protected tetraazine intermediate (73.9 mg, 29 μmol, 170%, 70% purity) as a pink solid. For C 87 H 98 N 25 O 20 + (M+H) + The calculated value of LCMS(ESI+) is 1812.7, and the measured value is 1813.0.

[0363] Alloc-protected tetrazine intermediate (30.8 mg, 1 Eq, 17.0 μmol) was dissolved in anhydrous DMF (3 mL), followed by the addition of tetra(triphenylphosphino)palladium (0) (1.96 mg, 218 μL, 7.8 mmol, 0.1 Eq, 1.70 μmol) and pyrrolidine (3.62 mg, 4.19 μL, 3 Eq, 51.0 μmol). After 10 minutes, the reaction mixture was purified by reversed-phase chromatography (10% → 100%, MeCN / water + 10 mM NH4HCO3) to give compound 53 as a pink solid (21.0 mg, 11 μmol, 63%, 86% purity). For C 78 H 86 N 25 O 20 + (M+H) + The calculated value of LCMS(ESI+) is 1692.6, and the measured value is 1692.9.

[0364] Compound 53 (21 mg, 86% Wt, 1 Eq, 11 μmol) was dissolved in anhydrous DMF (2 mL), followed by the addition of piperidine (18 mg, 21 μL, 20 Eq, 0.21 mmol). After 10 minutes, the desired product was precipitated by adding diethyl ether (10 mL). The precipitate was washed with diethyl ether (3 x 5 mL) and concentrated under vacuum to give a pink solid of the deprotected Fmoc intermediate (24.9 mg, 15 μmol, 140%, 89% purity). Assuming the yield was quantifiable, the product was used in the next step without any further purification. For C 63 H 76 N 25 O 18 + (M+H) + The calculated value of LCMS(ESI+) is 1470.6, and the measured value is 1470.9.

[0365] The deprotected intermediate of Fmoc (8.2 mg, 89% wt, 1 Eq, 5.0 μmol) was added to a solution of anhydrous DMF (500 μL) with DBCO-NHS ester (6.0 mg, 3 Eq, 15 μmol) and DIPEA (1.9 mg, 2.6 μL, 3 Eq, 15 μmol). After 45 min, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm), and the fraction containing the product was lyophilized to give compound 54 (4.1 mg, 1.8 μmol, 36%, 76% purity) as a pink solid. For C 82 H 87 N 26 O 20 - (MH) - The calculated value of LCMS(ESI-) is 1755.7, and the measured value is 1755.2. Example 27. Preparation of Compound 55

[0366] To a solution of compound 54 (8.00 mg, 1 Eq, 4.55 μmol) in anhydrous DMF (500 μL), 1-aminooctaethylene glycol (8.1 mg, 4.8 Eq, 22 μmol) was added, followed by the addition of HATU (6.5 mg, 3.8 Eq, 17 μmol) and DIPEA (3.53 mg, 4.76 μL, 6 Eq, 27.3 μmol). After 10 minutes, the desired product was precipitated by adding diethyl ether (10 mL). The precipitate was washed with diethyl ether (3 x 5 mL) and diluted with DMF (600 μL) and DMSO (300 μL). The reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm), and the fraction containing the product was lyophilized to give compound 55 (0.6 mg 0.2 μmol, 5%). For C 130 H 189 N 29 O 41 2+ (M / 2+H) + The calculated value of LCMS(ESI+) is 1406.7, and the measured value is 1406.8. Example 28. Preparation of Compound 57

[0367] Triethylamine (281 mg, 387 μL, 5 Eq, 2.78 mmol) was added to a solution of compound 6 (500 mg, 5.55 mL, 100 mmol, 1 Eq, 555 μmol) in DMF (5 mL), followed by Val-Ala-PABC (375 mg, 2.3 Eq, 1.28 mmol). The resulting solution was stirred for 4 hours and then purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give a diol intermediate as a white solid (431 mg, 356 μmol, 64.2%). For C 52 H 77 N 10 O 19 S2 + (M+H) + The calculated value of LCMS(ESI+) is 1209.5, and the measured value is 1210.1.

[0368] A solution of a diol intermediate (157 mg, 1 Eq, 130 μmol) in DMF (2 mL) was added to a mixture of bis(4-nitrobenzene) carbonate (118 mg, 3 Eq, 389 μmol), followed by the addition of DIPEA (83.9 mg, 113 μL, 5 Eq, 649 μmol). After stirring for 2 hours, an additional bis(4-nitrobenzene) carbonate (118 mg, 3 Eq, 389 μmol) was added to the reaction mixture. After a total of 4 hours of stirring, the reaction mixture was purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM) to give compound 56 (178 mg, 116 μmol, 89.1%) as a white solid. For C 66 H 83 N 12 O 27 S2 + (M+H) + The calculated value of LCMS (ESI+) is 1539.5, and the measured value is 1540.0.

[0369] Triethylamine (1.35 mg, 1.85 μL, 5 Eq, 13.3 μmol) and DMAP (650 μg, 5.32 μL, 1000 mmol, 2 Eq, 5.32 μmol) were added to a stock solution of compound 56 (4.10 mg, 26.6 μL, 100 mmol, 2 Eq, 5.32 μmol) in DMF, followed by a stock solution of paclitaxel (5.00 mg, 58.6 μL, 100 mmol, 2.2 Eq, 5.86 μmol) in DMF. The resulting solution was allowed to stand at room temperature for 25 hours and then purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 57 as a white solid (1.4 mg, 0.41 μmol, 16%, 88% purity). For C 148 H 176 N 12 O 49 S2 +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 1485.0, and the measured value is 1485.5. Example 29. Preparation of compound 59

[0370] To a solution of Fmoc-Val-Ala-PABC-PNP 4 (152.7 mg, 1.5 Eq, 224.3 μmol) in dry DCM (500 μL), a stock solution of mitomycin C in DMF (50.00 mg, 1.496 mL, 100 mmol, 1 Eq, 149.6 μmol), DIPEA (57.99 mg, 78.2 μL, 3 Eq, 448.7 μmol), and HOBt (34.35 mg, 224.3 μL, 1000 mmol, 1.5 Eq, 224.3 μmol) were added. After standing for 5 hours, the reaction mixture was purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM) to give a brown solid of Fmoc-protected intermediate (115 mg, 91.9 μmol, 62%, 70% purity). For C 46 H 53 N8O 11 + (M+NH4) + The calculated value of LCMS(ESI+) is 893.4, and the measured value is 894.0.

[0371] The Fmoc-protected intermediate (115 mg, 70% Wt, 1 Eq, 91.9 μmol) was dissolved in DMF (500 μL), and piperidine (78.3 mg, 90.8 μL, 10 Eq, 919 μmol) was added. The resulting solution was stirred for 1 hour and subsequently purified by silica gel rapid column chromatography (0% → 30% MeOH, in DCM) to give compound 58 (72.9 mg, 112 μmol, 121%) as a brown solid. For C 31 H 40 N7O9 + (M+H) + The calculated value of LCMS (ESI+) is 654.3, and the measured value is 654.8.

[0372] Compound 58 (5.0 mg, 70% Wt, 3 Eq, 5.4 μmol) was dissolved in DCM (100 μL), followed by the addition of compound 6 (1.6 mg, 18 μL, 100 mmol, 1 Eq, 1.8 μmol) and DIPEA (3.0 mg, 4.0 μL, 13 Eq, 23 μmol). The resulting solution was allowed to stand for 4 days and purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM). Subsequently, the crude product was purified by preparative HPLC (5% → 95%, MeCN / water, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 59 (3.4 mg, 1.7 μmol, 93%, 94% purity) as a brown solid. For C 84 H 108 N 18 O 31 S2 - M ·- The calculated value of LCMS(ESI-) is 1928.7, and the measured value is 1928.5. Example 30. Preparation of Compound 61

[0373] Add gemcitabine stock solution in DMF (50.00 mg, 1.900 mL, 100 mmol, 1 Eq, 190.0 μmol), DIPEA (73.66 mg, 99.3 μL, 3 Eq, 569.9 μmol), and DMAP (11.60 mg, 94.98 μL, 1000 mmol, 0.5 Eq, 94.98 μmol) to a solution of Fmoc-Val-Ala-PABC-PNP 4 (155.2 mg, 1.2 Eq, 228.0 μmol) in dry DCM (500 μL) to the solution. After standing for 3 hours, the reaction mixture was purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM), and the crude product was subsequently purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give a white solid Fmoc-protected intermediate (15 mg, 19 μmol, 9.8%). For C 40 H 43 F2N6O 10 + (M+H) + The calculated value of LCMS (ESI+) is 805.7, and the measured value is 805.3.

[0374] The Fmoc-protected intermediate (15 mg, 1 Eq, 19 μmol) was dissolved in DMF (200 μL), and triethylamine (57 mg, 78 μL, 30 Eq, 0.56 mmol) was added. The resulting solution was allowed to stand for 22 hours and then purified by silica gel rapid column chromatography (0% → 100% MeOH, in DCM) to give compound 60 (8.9 mg, 15 μmol, 82%) as a white solid. For C 25 H 33 F2N6O8 + (M+H) + The calculated value of LCMS(ESI+) is 583.2, and the measured value is 583.6.

[0375] Compound 60 (3.8 mg, 2.1 μmol, 42%) was dissolved in DMF (250 μL), followed by the addition of compound 6 (4.6 mg, 51 μL, 100 mmol, 1 Eq, 5.1 μmol) and triethylamine (5.2 mg, 7.1 μL, 10 Eq, 51 μmol). The resulting solution was allowed to stand for 22 hours and then purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 61 (3.8 mg, 2.1 μmol, 42%) as a white solid. For C 72 H 96 F4N 16 O 29 S2 +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 894.3, and the measured value is 895.0. Example 31. Preparation of compound 63

[0376] Sodium hydride (9.0 mg, 60% wt, 2.0 Eq, 224 μmol) was added to a solution of gefitinib (50.0 mg, 1.0 Eq, 112 μmol) cooled at 0 °C in 1.1 mL of dry DMF and 2.5 mL of dry DCM. The reaction mixture was stirred at 0 °C for 1 h and then allowed to warm to room temperature, after which compound 4 (114 mg, 1.5 Eq, 168 μmol) was added. The reaction mixture was stirred at room temperature for 21 h and then purified by silica gel rapid column chromatography (0→10% MeOH, in DCM) to give the Fmoc-protected intermediate (16.0 mg, 15 μmol, 14%, 95% purity). For C 53 H 56 ClFN7O9 - (M-H+HCOOH) - The calculated value of LCMS(ESI-) is 988.4, and the measured value is 988.8.

[0377] Et3N (46.7 mg, 64.3 μL, 30 Eq, 461 μmol) was added to a solution of the Fmoc-protected intermediate (16.0 mg, 95% wt, 1.0 Eq, 15.4 μmol) in dry DMF (200 μL). The reaction mixture was stirred at room temperature for 60 h and purified by silica gel rapid column chromatography (0→40% MeOH, in DCM) to give compound 62 (11.4 mg, 14 μmol, 93%, 96% purity). For C 38 H 46 ClFN7O7 - (M-H+HCOOH) - The calculated value of LCMS(ESI-) is 766.3, and the measured value is 766.7.

[0378] Compound 6 (4.3 mg, 1.0 Eq, 4.8 μmol) was added to a solution of compound 62 (11.4 mg, 96% Wt, 3.0 Eq, 14.3 μmol) in dry DMF (200 μL), followed by the addition of DIPEA (6.15 mg, 8.3 μL, 10 Eq, 47.6 μmol). The reaction mixture was stirred at room temperature for 20 h and subsequently purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 63 (4.4 mg, 1.8 μmol, 39%, 90% purity). For C 96 H 122 Cl2F2N 18 O 23 S2 2+ (M / 2+H) + The calculated value of LCMS(ESI+) is 1033.9, and the measured value is 1034.4. Example 32. Preparation of Compound 67

[0379] To a solution of paclitaxel (50.0 mg, 1.0 Eq, 58.6 μmol) in dry DMF (586 μL), bis(4-nitrobenzene) carbonate (35.6 mg, 2.0 Eq, 117 μmol) was added, followed by triethylamine (17.8 mg, 24.5 μL, 3.0 Eq, 176 μmol). The reaction mixture was stirred at 0 °C for 3 h and subsequently purified by silica gel rapid column chromatography (0→100% EtOAc / heptane) to give compound 64 as a white powder (32.9 mg, 30 μmol, 50%, 90% purity). For C 54 H55 N2O 18 + (M+H) + The calculated value of LCMS(ESI+) is 1019.3, and the measured value is 1019.7.

[0380] To a solution of compound 4 (750 mg, 1.0 Eq, 1.10 mmol) in dry THF (5.51 mL), tert-butyl (S)-methyl(pyrrolidone-2-ylmethyl)carbamate (394 mg, 1.7 Eq, 1.84 mmol) was added, followed by the addition of DIPEA (427 mg, 576 μL, 3.0 Eq, 3.31 mmol). The reaction mixture was stirred at room temperature for 3 hours and subsequently purified by silica gel rapid column chromatography (0→10% MeOH / DCM) to give a Boc-protected intermediate (861 mg, 1.14 mmol, 88%, 85% purity) as a pale yellow solid. For C 42 H 57 N6O8 + (M+NH4) + The calculated value of LCMS(ESI+) is 773.4, and the measured value is 773.7.

[0381] TFA (2.14 g, 1.43 mL, 20 Eq, 18.7 mmol) was added to a solution of a Boc-protected intermediate (833 mg, 85% Wt, 1.0 Eq, 937 μmol) in dry DCM (2.8 mL) at -15 °C. The reaction mixture was stirred at -15 °C for 4 h, followed by precipitation with Et₂O. The solid was collected by filtration, washed with more Et₂O, and concentrated under vacuum to give compound 65 (737 mg, 947 μmol, 101%, 99% purity) as a brown solid. For C 37 H 46 N5O6 + (M+H) + The calculated value of LCMS (ESI+) is 656.3, and the measured value is 656.6.

[0382] Compound 64 (29.3 mg, 90% wt, 1.3 Eq, 25.9 μmol) was added to a solution of compound 65 (20.2 mg, 99% wt, 1.0 Eq, 19.9 μmol) in dry DMF (100 μL) and dry DCM (200 μL), followed by the addition of DIPEA (7.7 mg, 10.4 μL, 3.0 Eq, 59.7 μmol). The reaction mixture was stirred at room temperature for 1 hour and purified by silica gel rapid column chromatography (0→5% MeOH / DCM) to give an Fmoc-protected intermediate (26.5 mg, 16.0 μmol, 78%, 90% purity) as a pale yellow solid. For C 85 H 95 N6O 21 + (M+H) + The calculated value of LCMS (ESI+) is 1535.7, and the measured value is 1536.4.

[0383] Piperidine (4.0 mg, 4.6 μL, 3.0 Eq, 46.6 μmol) was added to a solution of the Fmoc-protected intermediate (26.5 mg, 90% wt, 1.0 Eq, 15.5 μmol) in dry DMF (200 μL). The reaction mixture was stirred at room temperature for 1 hour and subsequently purified by silica gel rapid column chromatography (0→20% MeOH / DCM) to give compound 66 as a white solid (18.9 mg, 14.4 μmol, 93%, 100% purity). For C 70 H 85 N6O 19 + (M+H) + The calculated value of LCMS(ESI+) is 1313.6, and the measured value is 1314.1.

[0384] Compound 6 (1.4 mg, 15 μL, 1.0 Eq, 1.5 μmol) was added to a solution of compound 66 (5.0 mg, 2.5 Eq, 3.8 μmol) in dry DMF (90 μL), followed by the addition of Et3N (1.7 mg, 2.4 μL, 11.0 Eq, 17 μmol). The reaction mixture was stirred at room temperature for 30 h and then purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 67 (2.6 mg, 0.8 μmol, 53%, 100% purity). For C 162 H 196 N 16 O 51 S22- (M / 2-H) - The calculated value of LCMS(ESI-) is 1623.1, and the measured value is 1623.5. Example 33. Preparation of Compound 70

[0385] A solution of nemorubicin (150.0 mg, 1 Eq, 233.0 μmol) in DMF (1.2 mL) and DCM (2.0 mL) was cooled to 0 °C, followed by the addition of bis(4-nitrobenzene) carbonate (286.6 mg, 4.04 Eq, 942.1 μmol) and triethylamine (94.33 mg, 130 μL, 4 Eq, 932.2 μmol). After stirring at 0 °C for 4.5 hours, the reaction mixture was purified by silica gel rapid column chromatography (0% → 7% MeOH, in DCM) to give compound 68 (170.1 mg, 0.16 mmol, 68%, 75% purity) as a red oil. For C 39 H 41 N2O 17 + (M+H) + The calculated value of LCMS(ESI+) is 809.2, and the measured value is 809.5.

[0386] Compound 65 (63.3 mg, 75% Wt, 1 Eq, 63.0 μmol) and DIPEA (48.8 mg, 65.8 μL, 6 Eq, 378 μmol) were added to a solution of compound 68 (67.9 mg, 75% Wt, 1 Eq, 63.0 μmol) in anhydrous DMF (200 μL) and anhydrous DCM (500 μL). After stirring the reaction mixture at room temperature for 17 hours, piperidine (53.6 mg, 62.2 μL, 10 Eq, 630 μmol) was added. The reaction mixture was stirred at room temperature for 1 hour and subsequently purified by silica gel rapid column chromatography (0% → 30% MeOH, in DCM) to give compound 69 (48.2 mg, 43.7 μmol, 69.4%) as a red oil. For C 55 H 71 N6O 18 + (M+H) + The calculated value of LCMS(ESI+) is 1103.5, and the measured value is 1103.9.

[0387] A solution of compound 6 in DMF (1.9 mg, 21 μL, 100 mmol, 1.0 Eq, 2.1 μmol) was added to a solution of compound 69 (6.9 mg, 3 Eq, 6.3 μmol) in anhydrous DMF (150 μL), followed by the addition of triethylamine (1.1 mg, 1.5 μL, 5 Eq, 10 μmol). After stirring at room temperature for 22 hours, N,N-dimethyl-1,2-ethanediamine (0.08 mg, 0.1 μL, 0.4 Eq, 0.9 μmol) was added to quench unreacted and single-reacted compound 6. The reaction mixture was stirred for 15 minutes and then purified by silica gel rapid column chromatography (0% → 8% MeOH, in DCM) to give compound 70 (3.4 mg, 1.2 μmol, 58%) as a red oil. For C 132 H 172 N 16 O 49 S2 2+ (M / 2+H) + The calculated value of LCMS(ESI+) is 1415.0, and the measured value is 1415.4. Example 34. Preparation of Compound 72

[0388] The synthesis of compound 71 is described in PCT / EP2021 / 075401 (Example 7) (which is incorporated herein by reference). Compound 58 (18.2 mg, 1 Eq, 27.8 μmol) was dissolved in DMF (150 μL), and compound 71 (20 mg, 91.5% wt, 1.3 Eq, 35 μmol) (dissolved in 50 μL dry DMF) was added, followed by the addition of triethylamine (7.0 mg, 9.6 μL, 2.5 Eq, 69 μmol). The reaction mixture was allowed to stand for 20 h and then purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 72 (24.6 mg, 23.3 μmol, 83.7%, 98.5% purity) as a brown solid. For C 47 H 65 N 10 O 16 S + (M+NH4) + The calculated value of LCMS (ESI+) is 1057.4, and the measured value is 1057.8. Example 35. Preparation of compound 75

[0389] To a solution of β-lapaquinone (100 mg, 1.0 Eq, 405 μmol) in THF (3000 μL) and water (3000 μL), Na₂S₂O₄ (356 mg, 4.3 Eq, 1.74 mmol) was added, followed by TBAB (659 mg, 5.0 Eq, 2.02 mmol). The reaction mixture was heated to 80 °C. A solution of KOH (504 mg, 20 Eq, 8.09 mmol) in water (800 μL) was added, followed by a solution of Boc-p-aminobenzyl bromide (656 mg, 5.5 Eq, 2.23 mmol). After refluxing the reaction mixture for 150 min, ethyl acetate (10 mL) was added. The organic layer was washed with water (3 x 10 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was then dissolved in heptane (5 mL) and purified by silica gel rapid column chromatography (0→20% EtOAc, in heptane) to give a Boc-protected intermediate (123 mg, 203 μmol, 50%, 74% purity) as a pale yellow oil. For C 27 H 32 NO5 + (M+H) + The calculated value of LCMS(ESI+) is 450.2, and the measured value is 450.3.

[0390] TFA (500 μL) was added to a solution of the Boc-protected intermediate (123 mg, 74% Wt, 1.0 Eq, 203 μmol) cooled to 0 °C in 2000 μL of dry DCM. The reaction mixture was stirred at 0 °C for 3 h and then concentrated under vacuum to give compound 73 (94 mg, 203 μmol, 100%, 100% purity). The yield is assumed to be quantifiable. For C... 22 H 24 NO3 - (M+H) - The calculated value of LCMS(ESI-) is 350.1, and the measured value is also 350.1.

[0391] DIPEA (74 mg, 99 μL, 3.0 Eq, 568 μmol) and HATU (108 mg, 1.5 Eq, 284 μmol) were added to a solution of Fmoc-Val-Ala-OH (117 mg, 1.5 Eq, 284 μmol) in dry DMF (350 μL) and dry DCM (2400 μL). After stirring the reaction mixture for 30 min, a solution of compound 73 (88 mg, 1.0 Eq, 189 μmol) in dry DMF (750 μL) was added. The reaction mixture was stirred for 10 min and subsequently purified by silica gel rapid column chromatography (0→3% MeOH, in DCM) to give compound 74 (193 mg, 0.2 mmol, 110%, 79% purity) as a pale yellow oil. For C 45 H 48 N3O7 + (M+H) + The calculated value of LCMS(ESI+) is 742.4, and the measured value is 742.6.

[0392] To a solution of compound 74 (193 mg, 79% Wt, 1.0 Eq, 205 μmol) in dry DMF (2500 μL), piperidine (52 mg, 61 μL, 3.0 Eq, 616 μmol) was added. The reaction mixture was stirred for 1 h and purified by silica gel rapid column chromatography (0→15% MeOH / DCM) to give the deprotected intermediate of Fmoc (64 mg, 98 μmol, 47%, 78% purity). For C 30 H 36 N3O5 - (MH) - The calculated value of LCMS(ESI-) is 518.3, and the measured value is also 518.3.

[0393] Compound 6 (8.9 mg, 1.0 Eq, 9.9 μmol) was added to a solution of the deprotected Fmoc intermediate (31.2 mg, 78% wt, 4.9 Eq, 48.6 μmol) in dry DMF (200 μL), followed by the addition of Et3N (15.0 mg, 20.6 μL, 15 Eq, 148 μmol). The reaction mixture was stirred at room temperature for 49 h and subsequently purified by preparative HPLC (30% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C185 μm OBD column, 30 x 100 mm) to give compound 75 (5.2 mg, 3.1 μmol, 32%, 100% purity). For C 82 H 105 N 10 O 23S2 + (M+H) + The calculated value of LCMS (ESI+) is 1661.7, and the measured value is 1662.1. Example 36. Preparation of Compound 77

[0394] A solution of midostaurin (22.4 mg, 1 Eq, 39.3 μmol) in anhydrous THF (200 μL) was cooled to 0 °C, followed by the addition of sodium hydride (3.3 mg, 60% wt, 2.1 Eq, 83 μmol). After stirring at 0 °C for 20 min, compound 4 (53.6 mg, 2.01 Eq, 78.7 μmol) was added. The reaction mixture was stirred for 10 min, then quenched with water (50 μL), and stirred at 0 °C for 1 h. Piperidine (66.8 mg, 77.6 μL, 20 Eq, 785 μmol) was then added to the reaction mixture. After stirring at 0 °C for 2.5 h, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel rapid column chromatography (0% → 35% MeOH, in DCM). The residue was dissolved in DCM (500 μL) and precipitated by adding cold diethyl ether (1.5 mL). The precipitate was washed with cold diethyl ether (3 x 1.5 mL) and concentrated under vacuum to give compound 76 as a white solid (23.2 mg, 25 μmol, 64%, 96% purity). For C 51 H 52 N7O8 + (M+H) + The calculated value of LCMS(ESI+) is 890.4, and the measured value is 890.8.

[0395] Compound 6 (4.1 mg, 95% Wt, 1.0 Eq, 4.3 μmol) and triethylamine (2.20 mg, 3.03 μL, 5 Eq, 21.7 μmol) were added to a solution of compound 76 (11.6 mg, 3 Eq, 13.0 μmol) in anhydrous DMF (250 μL). After stirring at room temperature for 18 hours, the reaction mixture was diluted with DCM (2 mL) and purified by silica gel rapid column chromatography (0% → 10% MeOH, in DCM) to give compound 77 (6.4 mg, 2.3 μmol, 53%, 86% purity). For C 124 H 137 N 19 O 29 S2 +2 ((M+NH4+H) / 2) +The calculated value of LCMS(ESI+) is 1210.5, and the measured value is 1210.9. Example 37. Preparation of compound 79

[0396] Fmoc-Val-Ala-OH (40.0 mg, 1.5 Eq, 97.4 μmol) was dissolved in dry DMF (0.500 mL) and added to a stock solution of lenalidomide in DMF (16.8 mg, 325 μL, 200 mmol, 1 Eq, 65.0 μmol), followed by the addition of DIPEA (33.6 mg, 45.3 μL, 4 Eq, 260 μmol) and HATU (49.4 mg, 2 Eq, 130 μmol). The reaction mixture was stirred for 20 hours and subsequently purified by silica gel rapid column chromatography (0→10% MeOH, in DCM) to give compound 78 (23 mg, 35 μmol, 54%) as a colorless oil. For C 36 H 38 N5O7 + (M+H) + The calculated value of LCMS (ESI+) is 652.3, and the measured value is 652.6.

[0397] To a solution of compound 78 (23 mg, 1 Eq, 35 μmol) in DCM (1.0 mL), piperidine (60 mg, 70 μL, 20 Eq, 0.71 mmol) was added, and stirring was continued for 1 hour. The reaction mixture was purified by silica gel rapid column chromatography (0→30% MeOH, in DCM) to give a white solid deprotected intermediate of Fmoc (18 mg, 34 μmol, 95%, 80% purity). For C 21 H 26 N5O5 - (MH) - The calculated value of LCMS(ESI-) is 428.2, and the measured value is 428.3.

[0398] Compound 6 (8.390 mg, 1 Eq, 9.314 μmol) and triethylamine (9.424 mg, 13.0 μL, 10 Eq, 93.14 μmol) were added to a solution of the deprotected Fmoc intermediate (12.00 mg, 3 Eq, 27.94 μmol) in anhydrous DMF (279.4 μL). After stirring at room temperature for 16 hours, the deprotected Fmoc intermediate (6.000 mg, 1.5 Eq, 13.97 μmol) and triethylamine (4.712 mg, 6.49 μL, 5 Eq, 46.57 μmol) were added. After stirring at room temperature for 3 hours, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 79 (6 mg, 3 μmol, 30%, 80% purity). For C 64 H 85 N 14 O 23 S2 + (M+H) + The calculated value of LCMS (ESI+) is 1481.5, and the measured value is 1481.9. Example 38. Preparation of Compound 82

[0399] A solution of gemcitabine (50 mg, 2 Eq, 0.19 mmol) in DMF (0.95 mL) was cooled to -15 °C (ice-salt bath). Bis(4-nitrobenzene) carbonate (29 mg, 1 Eq, 95 μmol) and DIPEA (37 mg, 50 μL, 3 Eq, 0.28 mmol) were added. After stirring at -15 °C for 3 hours, compound 65 (88 mg, 1.2 Eq, 0.11 mmol) and DIPEA (37 mg, 50 μL, 3 Eq, 0.28 mmol) were added. The ice bath was removed, and after stirring at room temperature for 15 minutes, the reaction mixture was purified by silica gel rapid column chromatography (0→20% MeOH, in DCM) to give compound 80 (57.4 mg, 59.0 μmol, 62%, 97% purity) as a white solid. For C 47 H 55 F2N8O 11 + (M+H) + The calculated value of LCMS (ESI+) is 945.4, and the measured value is 945.8.

[0400] Piperidine (15.5 mg, 18.0 μL, 3 Eq, 182 μmol) was added to a solution of compound 80 (57.4 mg, 1 Eq, 60.7 μmol) in anhydrous DMF (607 μL). After stirring at room temperature for 1 hour, the reaction mixture was purified by preparative HPLC (10% → 60% MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 81 (13.2 mg, 18.3 μmol, 30%, 100% purity) as a white solid. For C 32 H 45 F2N8O9 + (M+H) + The calculated value of LCMS(ESI+) is 723.3, and the measured value is 723.6.

[0401] Compound 6 (3.32 mg, 1 Eq, 3.69 μmol) and triethylamine (3.73 mg, 5.14 μL, 10 Eq, 36.9 μmol) were added to a solution of compound 81 (8.00 mg, 3 Eq, 11.1 μmol) in anhydrous DMF (221 μL). After stirring at room temperature for 18 hours, the reaction mixture was purified by preparative HPLC (20% → 70% MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 82 (2.8 mg, 1.3 μmol, 35%, 95% purity). For C 86 H 120 F4N 20 O 31 S2 +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 1034.4, and the measured value is 1034.7. Example 39. Preparation of Compound 86

[0402] Sodium carbonate (0.15 g, 4 Eq, 1.5 mmol) and di-tert-butyl dicarbonate (88 mg, 89 μL, 1.1 Eq, 0.40 mmol) were added to a solution of gemcitabine (96 mg, 1 Eq, 0.36 mmol) in 1,4-dioxane (6.65 mL) and water (1.65 mL). After stirring at room temperature for 24 hours, an additional 40 mg, 41 μL, 0.5 Eq, 0.18 mmol of di-tert-butyl dicarbonate was added. After stirring at room temperature for 18 hours, an additional 40 mg, 41 μL, 0.5 Eq, 0.18 mmol of di-tert-butyl dicarbonate was added. After stirring at room temperature for 6 hours, water (20 mL) was added, and the reaction mixture was extracted with EtOAc (2 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by silica gel rapid column chromatography (20→100% acetone, in DCM) to give a white solid Boc-protected intermediate (83 mg, 0.23 mmol, 62%, 99% purity). For C 14 H 20 F2N3O6 + (M+H) + The calculated value of LCMS (ESI+) is 364.1, and the measured value is 364.4.

[0403] Di-tert-butyl dicarbonate (640 mg, 0.67 mL, 13 Eq, 2.9 mmol) was added to a solution of the Boc-protected intermediate (83 mg, 1 Eq, 0.23 mmol) in 1,4-dioxane (4.6 mL). After stirring at 37 °C for 24 hours, the reaction mixture was allowed to reach room temperature and stirred for another 72 hours. Water (20 mL) was added, and the reaction mixture was extracted with EtOAc (2 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by silica gel rapid column chromatography (0→20% acetone, in DCM) to give compound 83 as a white solid (93.3 mg, 201 μmol, 88%, 100% purity). For C 19 H 28 F2N3O8 + (M+H) + The calculated value of LCMS (ESI+) is 464.2, and the measured value is 464.5.

[0404] To a solution of compound 83 (21 mg, 1 Eq, 45 μmol) in MeCN (0.45 mL), triethylamine (14 mg, 19 μL, 3 Eq, 0.14 mmol) and bis(4-nitrobenzene) carbonate (28 mg, 2 Eq, 91 μmol) were added. After stirring at room temperature for 18 hours, the reaction mixture was purified by silica gel rapid column chromatography (0→10% MeOH, in DCM) to give compound 84 (28 mg, 43 μmol, 95%, 97% purity) as a yellow solid. For C 26 H 31 F2N4O 12 + (M+H) + The calculated value of LCMS(ESI+) is 629.2, and the measured value is 629.5.

[0405] A solution of compound 65 (18 mg, 73% wt, 1.0 Eq, 17 μmol) in anhydrous DCM (0.13 mL) and DIPEA (6.6 mg, 8.9 μL, 3.0 Eq, 51 μmol) were added to a solution of compound 84 (14 mg, 1.3 Eq, 22 μmol) in anhydrous DCM (0.13 mL). After stirring at room temperature for 1 hour, the reaction mixture was purified by silica gel rapid column chromatography (0 → 20% MeOH, in DCM) to give a white solid Fmoc-protected intermediate (25 mg, 18 μmol, 99%, 81% purity). For C 57 H 71 F2N8O 15 + (M+H) + The calculated value of LCMS(ESI+) is 1145.5, and the measured value is 1145.9.

[0406] Piperidine (4.6 mg, 5.3 μL, 3 Eq, 54 μmol) was added to a solution of the Fmoc-protected intermediate (21 mg, 1 Eq, 18 μmol) in anhydrous DMF (0.2 mL). After stirring at room temperature for 4 hours, the reaction mixture was purified by silica gel rapid column chromatography (0→20% MeOH, in DCM) to give compound 85 (9.6 mg, 10 μmol, 57%, 98% purity). For C 42 H 61 F2N8O 13 + (M+H) + The calculated value of LCMS(ESI+) is 923.4, and the measured value is 923.8.

[0407] A solution of compound 85 (9.6 mg, 1 Eq, 10 μmol) in DCM (0.16 mL) was cooled to 0 °C, and TFA (52 μL, 68 Eq, 0.68 mmol) was added. After stirring at 0 °C for 3 hours, the reaction mixture was concentrated under vacuum to give a deprotected Boc intermediate (12.9 mg, 7.9 μmol, 76%, 58% purity) as a grayish-white solid. For C 32 H 45 F2N8O9 + (M+H) + The calculated value of LCMS(ESI+) is 723.3, and the measured value is 723.6.

[0408] Triethylamine (11 mg, 15 μL, 10 Eq, 0.10 mmol) and compound 6 (8.2 mg, 1.5 Eq, 16 μmol) were added to a solution of the deprotected Boc intermediate (9.9 mg, 1 Eq, 10 μmol) in anhydrous DMF (0.21 mL). After stirring at room temperature for 18 hours, triethylamine (11 mg, 15 μL, 10 Eq, 0.10 mmol) was added. After stirring at room temperature for 6 hours, HOBt (1.6 mg, 1 Eq, 10 μmol) was added. After stirring at room temperature for 18 hours, DMAP (1.3 mg, 1 Eq, 10 μmol) was added, and the reaction mixture was heated to 40 °C. After stirring at 40°C for 6 hours, the reaction mixture was purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) and then purified a second time by preparative HPLC (10% → 60%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 86 (1.2 mg, 1.1 μmol, 10%, 100% purity). For C 48 H 67 F2N 10 O 16 S + (M+H) + The calculated value of LCMS(ESI+) is 1109.4, and the measured value is 1109.8. Example 40. Preparation of Compound 87

[0409] To a solution of compound 56 (10.5 mg, 1.0 Eq, 6.8 μmol) in dry DMF (270 μL), HCl salt of daunorubicin (9.6 mg, 170 μL, 2.5 Eq, 17 μmol) was added, followed by the addition of Et3N (6.9 mg, 9.5 μL, 10 Eq, 68 μmol) and DMAP (0.8 mg, 1.0 Eq, 6.8 μmol). The reaction mixture was stirred at room temperature for 22 h and subsequently purified by preparative HPLC (5% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 87 (5.7 mg, 2.5 μmol, 36%, 100% purity). For C 108 H 128 N 12 O 41 S2 2- (M / 2-H) - The calculated value of LCMS(ESI-) is 1156.4, and the measured value is 1156.8. Example 41. Preparation of Compound 88

[0410] Compound 66 (8.43 mg, 1.0 Eq, 6.42 μmol) and triethylamine (1.95 mg, 2.68 μL, 3.0 Eq, 19.3 μmol) were added to a solution of compound 71 (4.79 mg, 1.3 Eq, 8.34 μmol) in dry DMF (100 μL). The reaction mixture was allowed to stand at room temperature for 8 hours, then diluted with DMF (500 μL) and purified by preparative HPLC (injection volume 650 μL DMF; 30% → 95%, MeCN / water + 1% AcOH, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm). The crude product was redissolved in 400 μL DMF and then purified by preparative HPLC (injection volume 450 μL DMF; 30% → 95%, MeCN / water + 1% AcOH, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to obtain compound 88 (3.5 mg, 2.0 μmol, 31%, 97.77% purity). For C 86 H 107 N8O 26 S + (M+H) + The calculated value of LCMS(ESI+) is 1699.7, and the measured value is 1700.2. Example 42. Preparation of Compound 91

[0411] Add 3-[(2R)-pyrrolidine-2-yl]propionate (23.1 mg, 2.5 Eq, 129 μmol) and DIPEA (19.9 mg, 26.9 μL, 3.0 Eq, 154 μmol) to a solution of Fmoc-Val-Ala-PABC-OPNP 4 (35.0 mg, 1.0 Eq, 51.4 μmol) in dry THF (2.57 mL). Stir the reaction mixture at room temperature under a nitrogen atmosphere. After 40 minutes, add 300 μL of dry DMF to aid dissolution. After 6.5 hours, concentrate the reaction mixture under vacuum to obtain a clear, pale yellow oil. The crude product was redissolved in DCM (3 ml), and the solution was purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give an unactivated acid intermediate as a pale yellow solid (47.4 mg, 60.2 μmol, 117%, 86.93% purity). For C 38 H 45 N4O8 + (M+H) + The calculated value of LCMS (ESI+) is 685.3, and the measured value is 685.8.

[0412] Add 3-(((ethylimino)methylene)amino)-N,N-dimethylpropane-1-amine hydrochloride (15.0 mg, 1.3 Eq, 78.2 μmol) and 2,3,5,6-tetrafluorophenol (13.0 mg, 1.3 Eq, 78.2 μmol) to a solution of the unactivated acid intermediate (47.4 mg, 1.0 Eq, 60.2 μmol) in dry DCM (661 μL). Add 50 μL of dry DMF to aid dissolution. Stir the reaction mixture at room temperature for 2 hours and concentrate under vacuum. Redissolve the crude product in DCM (1 mL) and purify by silica gel rapid column chromatography (0% → 30% acetone, in DCM) to give compound 89 (60 mg, 31 μmol, 52%, 43.64% purity). For C 44 H 48 F4N5O8 + (M+NH4) + The calculated value of LCMS(ESI+) is 850.3, and the measured value is 850.7.

[0413] Paclitaxel (34.0 mg, 1.25 Eq, 39.0 μmol) was added to a solution of compound 89 (60.0 mg, 1.0 Eq, 31 μmol) in a mixture of dry DCM (187 μL) and dry DMF (100 μL). After stirring the reaction mixture for 50 min, DMAP (4.2 mg, 1.1 Eq, 35.0 μmol) was added. After 30 min, the reaction mixture was diluted with DCM (3 mL) and purified by silica gel rapid column chromatography (0% → 20% MeOH, in DCM) to give the Fmoc-protected intermediate (49.3 mg, 26.5 μmol, 84%, 81.77% purity). For C 85 H 94 N5O 21 + (M+H) + The calculated value of LCMS (ESI+) is 1520.6, and the measured value is 1521.2.

[0414] Piperidine (6.77 mg, 7.86 μl, 3.0 Eq, 79.5 μmol) was added to a solution of the Fmoc-protected intermediate (49.3 mg, 1.0 Eq, 26.5 μmol) in DMF (200 μL). The reaction mixture was stirred at room temperature for 30 min and then diluted with DCM (4 mL). The solution was then purified by silica gel rapid column chromatography (0% → 15% MeOH, in DCM) to give compound 90 (15.7 mg, 11.9 μmol, 45%, 98.65% purity). For C 70 H 84 N5O 19 + (M+H) + The calculated value of LCMS (ESI+) is 1298.6, and the measured value is 1299.0.

[0415] Compound 6 (1.78 mg, 1.0 Eq, 1.98 μmol) and triethylamine (1.0 mg, 1.38 μL, 5.0 Eq, 9.88 μmol) were added to a solution of compound 90 (7.8 mg, 3.0 Eq, 5.93 μmol) in dry DMF (100 μL). The reaction mixture was allowed to stand at room temperature for 7.5 h, then maintained at -20 °C for 17 h. The reaction was then heated to room temperature and maintained for another 5 h, followed by dilution with DMF (500 μL) and subsequent purification by preparative HPLC (injection volume 550 μL DMF; 30% → 95%, MeCN / water + 1% AcOH, run time 23 min, Xbridge prep C18 5 μm OBD column, 30 x 100 mm) to give compound 91 (3.3 mg, 1.0 μmol, 52%, 100% purity). For C 162 H 198 N 14 O 51 S2 +2 (M / 2+H) + The calculated value of LCMS(ESI+) is 1609.1, and the measured value is 1609.6. Example 43. Preparation of compound 92

[0416] Compound 90 (7.85 mg, 1.0 Eq, 5.96 μmol) in dissolved dry DMF (70 μl) was added to a solution of compound 71 (4.45 mg, 1.3 Eq, 7.75 μmol) in dry DMF (30 μL), followed by triethylamine (1.81 mg, 2.49 μL, 3.0 Eq, 17.9 μmol). The reaction mixture was allowed to react at room temperature for 7 hours, and then cooled to -20 °C for 17 hours. The reaction was then heated to room temperature and maintained for 5 hours, followed by dilution with DMF (400 μl) and subsequent purification by preparative HPLC (550 μL DMF injection volume; 30% → 95%, MeCN / water + 1% AcOH, run time 23 min, Bridge Prep C18 5 μm OBD column, 30 x 100 mm) to give compound 92 (6.6 mg, 3.8 μmol, 64%, 97.87% purity). For C 86 H 106 N7O 26 S + (M+H) + The calculated value of LCMS (ESI+) is 1684.7, and the measured value is 1685.1. Example 44. Preparation of compound 96

[0417] CSI (147 mg, 90 μL, 1.2 Eq, 1.1 mmol) was added to a solution of 2-(Fmoc-amino)ethanol (250 mg, 1.0 Eq, 882 μmol) cooled to -35 °C in dry DCM (20 mL). After stirring at -35 °C for 20 min, Et3N (268 mg, 369 μL, 3.0 Eq, 2.7 mmol) was added, followed by 2-(2-aminoethoxy)ethanol-1-ol (111 mg, 106 μL, 1.2 Eq, 1.1 mmol). The reaction mixture was warmed to room temperature and stirred for 1 h. PNP chloroformate (267 mg, 1.5 Eq, 1.3 mmol) was added, followed by another Et3N (268 mg, 369 μL, 3.0 Eq, 2.7 mmol). The reaction mixture was stirred at room temperature for 1 hour, concentrated under vacuum, dissolved in DCM (6 mL), and purified by silica gel rapid column chromatography (0% → 3% MeOH, in DCM) to give compound 93 (387 mg, 560 μmol, 63%, 95% purity). For C 29 H 31 N4O 12 S + (M+H) + The calculated value of LCMS (ESI+) is 659.2, and the measured value is 659.3.

[0418] Compound 94 (50.0 mg, 1.0 Eq, 20.8 μmol, commercially available from Levena Biopharma) was added to a solution of compound 93 (20.6 mg, 95% Wt, 1.5 Eq, 31.3 μmol) in dry DCM (400 μL) and dry DMF (200 μL), followed by the addition of DIPEA (26.9 mg, 36.2 μL, 10 Eq, 208 μmol) and HOBt (3.2 mg, 1.0 Eq, 20.8 μmol). After stirring at room temperature for 20 hours, piperidine (17.7 mg, 20.5 μL, 10 Eq, 208 μmol) was added. The reaction mixture was stirred for 3 hours and purified by silica gel rapid column chromatography (0% → 30% MeOH, in DCM) to give compound 95 (48.8 mg, 18.1 μmol, 87%, 100% purity) as a white solid. For C 130 H 212 N24O 35 S 2+ (M / 2+H) + The calculated value of LCMS(ESI+) i...

Claims

1. A method for preparing antibody payload conjugates with a DAR of 6 or higher, the method comprising: (a) Provides a structure Ab(F) 1 ) z The modified antibody, where Ab is an antibody, z is 2 or 4, and F 1 It is a click probe; (b) Mix the modified antibody with z equivalents of Q 1 L A (F 2 ) y The reaction, in which Q 1 It is for F 1 Reactive click probe, L A It is a heterofunctional (y+1) valence linker, where y is 2, 3, or 4, and F 2 It is for Q 1 Non-reactive click probes to obtain structures with Ab(Z) 1 L A (F 2 ) y ) z The antibody-linker construct, in which Z 1 Through F 1 and Q 1 Linking groups obtained from the reaction; (c) Make the antibody-linker construct with a z×y equivalent of Q 2 (L B )D x The reaction, in which Q 2 It is for F 2 Reactive click probe, L B It is a (x+1) valence linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is the effective load molecule, to obtain the structure Ab(Z). 1 L A (Z 2 (L B )D x ) y ) z Coupled with Z 2 Through F 2 and Q 2 Linking groups obtained from the reaction.

2. The method according to claim 1, wherein y is 2, and L A It is a heterobifunctional trivalent linker with the following structure: -(L 11 )-BM(L 12 -)(L 13 -) in: -L 11 With Q 1 or Z 1 Connect, and L 12 and L 13 With F 2 or Z 2 Connection, where L 11 L 12 and L 13 Each is individually composed of one or more structural units selected from the following: CH2, CH=CH, C≡C, C(O), NR 13 O, S, S(O), S(O)2, PR 13 and P(O)R 13 Preferably, these structural units are selected from CH2, CH=CH, C(O), and NR. 13 , O, S, S(O) and S(O)2, and -BM is a branch portion, preferably selected from carbon atom, nitrogen atom, phosphorus atom, (hetero)aromatic ring, (hetero)ring or polycyclic portion, more preferably BM is nitrogen atom.

3. The method according to claim 1 or 2, wherein x is 2, and L B It is a heterobifunctional trivalent linker with the following structure: -(L 1 )-[(L 2 ) o -(L 3 ) p -(L 4 ) q -]2 in: -L 1 With Q 2 or Z 2 Connect, and the L appears twice. 4 All are connected to D; -L 1 L 2 L 3 and L 4 Each individually is Q 2 or Z 2 Connector that is connected to D; -o, p, and q are each individually 0 or 1, preferably where o = p = 1; And preferably, wherein: (a) Connector L 1 It is represented by the following: -(W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A) d’ -(B) e '-(A) f’ -(C(O)) g '-]2, in: -d and d' are either 0 or 1 individually; -e and e' are individually integers in the range of 1-10; -f and f' are either 0 or 1 individually; -g and g' are individually integers in the range of 0-10; -k = 0 or 1, provided that if k = 1, then d = 0; -A is an aminocarbamoyl or acylsulfonamide group based on structure (23). Where a = 0 or 1, and R 13 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, these C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S, and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 D is connected to N via a spacer portion, preferably wherein the spacer portion is -(B). g -(C(O)) g -(L 2 ) o -(L 3 ) p -(L 4 ) q -; -W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH2) m C(O)-、-C(O)(CH2) m C(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, where m is an integer in the range of 0-10; -B is a part of -CH2-CH2-O- or -O-CH2-CH2-, or (B) e It is -(CH2-CH2-O) e1 -CH2-CH2- part, where e1 is an integer in the range of 1-10; -BM is the branch part, two -(A) d -(B) e -(A) f -(C(O)) g - Connect it, and both (C(O)) g’ Some of them are related to -(L) 2 ) o -(L 3 ) p -(L 4 ) q -D connects, where L 2 L 3 L 4 o, p, q, and D are each selected individually; and / or (b) Connector L 2 It is a peptide spacer, preferably a peptide spacer containing 1-5 amino acids, and more preferably a dipeptide, tripeptide or tetrapeptide spacer; and / or (c) Connector L 3 It is a self-destructing spacer, preferably: - When o = 1, L 3 It is a p-aminobenzyloxycarbonyl (PABC) derivative based on structure (26): - When o = 1, L 3 It is a glucuronide derivative based on structure (27): Where R 21 It is H, R 26 or C(O)R 26 , where R 26 It is C1-C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl group, C3-C 10 alkyl(hetero)aryl groups and C3-C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally selected from O, S, and NR. 28 One or more heteroatoms are interrupted, where R 28 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably wherein R 21 Is it H or C(O)R 26 , where R 26 = 4-N-methylpiperazine or morpholine, most preferably wherein R 21 It is H; and / or (d) Connector L 4 The spacers are selected from the following: -Based on structure-NR 22 -(C x -alkylene)-C(O)-aminoalkanoic acid spacers, where x is an integer in the range of 1-20, and R 22 It is H or C1-C4 alkyl; or -Based on structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7 -C(O)- ethylene glycol spacer, where e6 is an integer in the range of 1-10, e7 is an integer in the range of 1-3, and R 22 It is H or C1-C4 alkyl; or -Based on structure-NR 22 -(C x -alkylene)-NR 22 -(C(O)) h - a diamine spacer, where h is 0 or 1, x is an integer in the range of 1-10, and R 22 It is H or C1-C4 alkyl.

4. The method according to any one of claims 1-3, wherein the reactions in step (b) and step (c) are both cycloadditions, preferably wherein the reaction in step (b) is a 1,3-dipolar cycloaddition and the reaction in step (c) is an anti-electron demand Diels-Alder cycloaddition.

5. The method according to any one of claims 1-4, wherein: -Q 1 and Q 2 Both are individually click probes comprising a (hetero)cyclic alkyne moiety or a (hetero)cyclic -E-olefin moiety, preferably wherein these click probes are selected from the group consisting of (Q2)-(Q20) according to the following structures: in: - Tilde key indicates L A or L B It can be connected to any available carbon or nitrogen atom; The nitrogen atoms in (Q10), (Q13), (Q14), and (Q15) can react with L A or L B Connected, or containing hydrogen atoms or being substituted; -B (-) It is an anion; -B (+) It is a cation; and / or -F 1 and F 2 Both are individually click probes selected from the group consisting of: azides, tetrazides, triazines, nitrones, nitriles, nitriles, nitrile imines, diazo compounds, dioxothiophene, syldiphenyl ketones, iminosyldiphenyl ketones, and catechols, preferably wherein these click probes are selected from the group consisting of (F1)-(F10) according to the following structures: in: - The tilde key indicates an interaction with Ab or L. A The connection, where for (F3), (F4), (F8), and (F9), can be made via any of these wavy bonds, and the other wavy bonds can then be connected to groups selected from: hydrogen, C1-C... 24 Alkyl groups, C2-C 24 Acyl group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 Sulfonyl groups, which may be optionally substituted and may be optionally selected from O, S, and NR. 32 One or more heteroatoms are interrupted, where R 32 It is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

6. The method according to any one of claims 1-5, wherein: -F 1 It is an azide or nitroketone, and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q17), (Q18), (Q19), and (Q19a) as defined in claim 5; and -F 2 It is tetrazine, and Q 2 It is a dicyclic nonyne or a cyclic olefin, preferably wherein Q 2 It is according to (Q8), (Q44), (Q47), (Q48), (Q49), (Q54), (Q55), or (Q56) as defined in claim 5, and F 2 It is based on (F8a): in: -R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl, preferably wherein R 29 It is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl.

7. The method according to any one of claims 1-6, wherein the payload D is a pharmaceutically active compound, preferably a cytotoxin.

8. The method according to any one of claims 1-7, wherein the antibody provided in step (a) is based on the structure: Ab[(L 6 )S(F 1 )] z , in: -z is 2 or 4; -L 6 Yes - GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - where G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fuc, w is 0 or 1, w' is 0 or 1, and L 7 It is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-.

9. A coupling compound that can be obtained by the method according to any one of claims 1-8.

10. A coupling having the structure Ab(Z) 1 L A (Z 2 (L B )D x ) y ) z ,in: -Ab is an antibody; -Z 1 and Z 2 It is a linker group that can be obtained through a reaction between two click probes; -L A It is a heterofunctional (y+1) valence linker; -L B It is a (x+1) valence connector; -x is 1, 2, 3 or 4; -y is 2, 3 or 4, provided that x+y is at least 4; -z is 2 or 4; -D represents the payload.

11. The coupling according to claim 10, wherein Z 1 and Z 2 Selected separately from structures (Z2)-(Z38a): in: - Tilde key indicates L A or L B It can be connected to any available carbon or nitrogen atom; - Ring Z with Ab or L A The linker is preferably selected from triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, oxazoline, isoxazoline, pyrazoline, piperazine or pyridazine; Nitrogen atoms in (Z10), (Z13), (Z14), and (Z15) can react with L A or L B Connected, or containing hydrogen atoms or being substituted; -B (-) It is an anion; -B (+) It is a cation.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a conjugate according to any one of claims 9-11.

13. A connector-constructor having a structure Q 1 L A (F 2 ) y ,in: -Q 1 It is a click probe; -L A It is a heterofunctional (y+1) valence linker; -F 2 It is for Q 1 Non-reactive click probes; -y is 2, 3 or 4.

14. The connector-construction of claim 13, wherein: -Q 1 It is a benzocyclocyclic or tetramethylated (hetero)cyclic alkyne, preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q17), (Q18), (Q19) and (Q19a) as defined in claim 5; -F 2 It is a tetrazine, preferably according to (F8a): in: -R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl, preferably wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl; and -y is 2, 3 or 4, preferably y is 2 or 3, and most preferably y is 2.

15. A method for the following: - Targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting the conjugate according to any one of claims 9-11 with cells that may express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor; and / or - A method for treating cancer, comprising administering to a subject in need the conjugate according to any one of claims 9-11, wherein cancer cells specifically express extracellular receptors. Preferably, the extracellular receptor is selected from the group consisting of: 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, short proteoglycan, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / glucan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38. CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, Sealin-18.2, Sealin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, Fibronectin, FLT3, Folate receptor α, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylate cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, connexin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tendinin, or antibodies specific to extracellular proteins caused by viral infection and / or to tumor-associated carbohydrate antigens (TACA).

Images