Antibody-drug conjugates with masked PBD dimers

Abstract

The present invention relates to conjugates of masked PBD dimers suitable for the treatment of cancer. The inventors have found that linking the masked PBD dimers via the glycans of a cell-binding agent increases the efficacy and tolerability of the PBD-payload. These findings allow for more effective cancer treatment with fewer adverse side effects. Thus, the present invention relates to conjugates of structure (1): AB-[L-(D) x ] y (1) where AB is a cell binding agent; x is 1 or 2; y is 1 or 2; D is a masked PBD dimer payload; and L is a linker connecting AB to D.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to the field of medicine. More particularly, the present invention relates to a masked pyrrolobenzodiazepine dimer and an antibody-drug conjugate prepared therewith, in particular an antibody-drug conjugate for the treatment of cancer based on the masked pyrrolobenzodiazepine dimer for release in the tumor microenvironment.

[0002] [background] Antibody-drug conjugates (ADCs), considered one of the major classes of targeted therapies, consist of antibodies to which pharmaceutical agents are attached. Antibodies (also known as binders or ligands) can be small protein formats (e.g., scFv, Fab fragments, DARPins, affibodies, etc.), but are generally IgG-type monoclonal antibodies (mAbs), selected for their high selectivity and affinity for a given antigen, their long circulating half-life, and little to no immunogenicity. Thus, mAbs, as ligands for carefully selected biological receptors, provide an ideal targeting platform for the selective delivery of pharmaceuticals. 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 finally the release of active catabolites. The cytotoxic agent can be in other formats, such as small molecule toxins, protein toxins, or oligonucleotides. As a result, tumor cells can be selectively eradicated while sparing normal cells not targeted by the antibody. Similarly, chemical conjugation of antimicrobial agents (antibiotics) to antibodies can be applied to treat bacterial infections, while conjugates of anti-inflammatory drugs are under investigation for the treatment of autoimmune diseases, and the attachment of oligonucleotides to antibodies, for example, is a potentially promising approach for the treatment of neuromuscular diseases. Thus, the concept of targeted delivery of active pharmaceutical agents to optimal specific cellular locations is a powerful approach for the treatment of a wide range of diseases, with many beneficial aspects relative to the systemic delivery of the same drugs.

[0003] ADCs are prepared by conjugation of a linker-drug to a protein, a process known as bioconjugation. See GT Hermanson, "Bioconjugate Techniques," Elsevier, 3, incorporated by reference. rdMany techniques for bioconjugation are known, as summarized in Ed. 2013. Conceptually, the method for preparing an ADC by bioconjugation involves the reaction of x reactive moieties F present on an antibody with complementary reactive moieties Q (payload) present on a pharmaceutical agent (see Figure 1).

[0004] Typically, a chemical linker is present between Q and the payload. This linker must have several important attributes, including the need to be stable in plasma after prolonged drug administration. A stable linker allows localization of the ADC to the expected site or cell in the body and prevents premature release of the payload in the circulation, which could indiscriminately induce any kind of undesirable biological response, thereby reducing the therapeutic index of the ADC. Upon internalization, the ADC must be processed to ensure effective release of the payload so that it can exert its mode of action within the cell. The linker may also contain a spacer element. Two families of linkers exist: non-cleavable and cleavable. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload that is completely stable under physiological conditions, regardless of which organ or biological compartment the antibody-drug conjugate is present in. As a result, release of the payload from an ADC with a non-cleavable linker depends on complete (lysosomal) degradation of the antibody after internalization of the ADC into the cell. As a result of this degradation, the payload will be released, still bearing the linker and peptide fragments and / or amino acids from the antibody to which the linker was originally attached. Cleavable linkers exploit the intrinsic properties of cells or cellular compartments for the selective release of the payload from the ADC, generally leaving no trace of the linker after processing. There are three commonly used mechanisms for cleavable linkers: (1) sensitivity to specific enzymes, (2) pH sensitivity, and (3) sensitivity to the redox state of the cell (or its microenvironment). Cleavable linkers may also contain self-immolative units based, for example, on para-aminobenzyl alcohol groups and their derivatives. Linkers may also contain additional elements, often referred to as spacer or stretcher units, to connect the linker to a reactive group for attachment to an antibody via a reactive moiety F present on the antibody.

[0005]

[0005] The reactive group F may be naturally occurring in an antibody, for example the reactive moiety may be a lysine or cysteine ​​side chain that can be used for acylation (lysine side chain) or alkylation (cysteine ​​side chain).

[0006]

[0006] Acylation of the e-amino group of the lysine side chain is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, e.g., SMCC, as applied in the production of Kadcyla®.

[0007]

[0007] Various reagents are known for alkylating thiol groups in cysteine ​​side chains (see Figure 2). Among cysteine ​​alkylation methods, most are based on the use of maleimide reagents, as applied, for example, in the production of Adcetris®, Polivy®, and Padcev®. In addition to standard maleimide derivatives, various maleimide variants are also applied for more stable cysteine ​​conjugation, as demonstrated, for example, by James Christie et al., J. Contr. Rel. 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol. 2014, 32, 1059-1062 (both incorporated by reference).Other approaches for cysteine ​​alkylation include various approaches based on nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem. 2008, 19, 759-765, which are incorporated by reference), or nucleophilic addition at unsaturated bonds, such as reaction with acrylate reagents (see, e.g., 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), reaction with phosphonamidates (see, e.g., Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, which are incorporated by reference), reaction with allenamides (see, e.g., Abbas et al., Bioconj. Chem. 2019, 58, 11625-11630, which are incorporated by reference), and the like. al., Angew. Chem. Int. Ed 2014, 53, 7491-7494), reaction with cyanoethynyl reagents (see, e.g., Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, which is incorporated by reference), reaction with vinyl sulfones (see, e.g., Gil de Montes et al., Chem. Sci. 2019, 10, 4515-4522, which is incorporated by reference), or reaction with vinyl pyridines (see, e.g., Seki et al., Chem. Sci., 2021, 12, 9060-9068 and https: / / iksuda.com / science / permalink / (accessed July 26, 2020)).An alternative approach to antibody conjugation without antibody re-engineering involves the reduction of interchain disulfide bridges followed by the use of bis-sulfone reagents (see, e.g., Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both of which are incorporated by reference), mono- or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., J. Am. Chem. Soc. 2010, 132, 1960-1965, both of which are incorporated by reference), or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., J. Am. Chem. Soc. 2010, 132, 1960-1965, both of which are incorporated by reference). al., Org. Biomol. Chem. 2014, 37, 7261-7269), bis-maleimide reagents (see, e.g., WO 2014114207), bis(phenylthio)maleimides (see, e.g., 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), bis-bromopyridazinediones (see, e.g., Robinson et al., RSC Advances 2017, 7, 9073-9077, which are incorporated by reference), bis(halomethyl)benzenes (see, e.g., Ramos-Tomillero et al., RSC Advances 2017, 7, 9073-9077, which are incorporated by reference), and the like. These include the addition of payloads linked to cysteine ​​cross-linking reagents such as cysteine ​​cross-linkers (see, e.g., WO 2013173391), bis(halomethyl)aromatic compounds (see, e.g., WO 2013173391). Typically, ADCs prepared by cysteine ​​cross-linking have a drug-antibody loading (DAR4) of about 4. Another useful technique for conjugation to cysteine ​​side chains is via disulfide bonds, which are bioactivatable linkages that have been utilized to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci. 2017, 8, 366-370, which is incorporated by reference).

[0008] In addition to conjugation to the side chains of the naturally occurring amino acids lysine or cysteine, various other conjugation techniques have been explored based on a two-step approach involving (a) the introduction of a new reactive group F, followed by (b) reaction with another complementary reactive group Q. For example, one method can be used to introduce a given number of reactive moieties F, which can be two, four, or eight, into an antibody (see Figure 3).

[0009]

[0009] Examples of non-natural reactive functional groups F that can be used in linker-drug bioconjugation are oxime groups suitable for oxime ligation or azide groups suitable for click chemistry conjugation. Oximes can be introduced into antibodies by genetic encoding of non-natural amino acids, for example, p-acetophenylalanine, as shown in Axup et al. Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, which is incorporated by reference, or by enzymatic alkylation of a cysteine ​​present in the CAAX sequence with a prenyl group containing a remote keto group, as disclosed in WO2012153193. Azides can be introduced into antibodies by genetically encoding p-azidomethylphenylalanine or p-azidophenylalanine, as demonstrated, for example, by Axup et al., Proc. Nat. Acad. Sci. 2012, 109, 16101-16106. Similarly, Zimmerman et al., Bioconj. Chem. 2014, 25, 351-361, incorporated by reference, used cell-free protein synthesis to introduce p-azidomethylphenylalanine (AzPhe) into monoclonal antibodies for conversion to ADCs via metal-free click chemistry. Also, Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097, incorporated by reference, demonstrated that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins using auxotrophic bacteria and further converted to protein conjugates via click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUAGenetic encoding of aliphatic azides in recombinant proteins using pairs was demonstrated by Nguyen et al., J. Am. Chem. Soc. 2009, 131, 8720-8721, which is incorporated by reference, and labeling was achieved by click chemistry via either copper-catalyzed alkyne azide cycloaddition (CuAAC) or strain-promoted alkyne azide cycloaddition (SPAAC). In addition to CuAAC and SPAAC, bioconjugation of linker-drugs to antibodies (and other biomolecules such as glycans and nucleic acids) can be achieved by various other metal-free click chemistries. See, for example, Nguyen and Prescher, Nature Rev. Chem. 2020, 4, 476-489, which is incorporated by reference. For example, oxidation of specific tyrosines in proteins can yield ortho-quinones, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes. See, for example, Bruins et al., Chem. Eur. J. 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. 2020, 11, 9011-9016, which is incorporated by reference. The tetrazine moiety can also be introduced into proteins or glycans by various means, e.g., genetic encoding or chemical acylation, and can also undergo cycloaddition with cyclic alkenes and alkynes. A list of pairs of functional groups F and Q for metal-free click chemistry is shown in Figure 4.

[0010] In SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne, and cycloaddition with an azide-modified antibody is facilitated by the relief of ring strain. Conversely, the linker-drug can be functionalized with an azide, and the antibody can be functionalized with a cyclic alkyne. Various strained alkynes suitable for metal-free click chemistry are shown in Figure 5.

[0011]

[0011] A method gaining popularity in the field of ADCs is based on the enzymatic introduction of a non-natural functional group F. For example, Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871, incorporated by reference, uses the bacterial enzyme transglutaminase (BTG or TGase) to introduce an azide moiety onto an antibody. A genetic method based on C-terminal TGase-mediated azide introduction followed by conversion in ADCs via metal-free click chemistry was reported by Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0012]

[0012] In International Publication No. 2014065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12, and Wijdeven at al. MAbs 2022, 14, 2078466 (all incorporated by reference) have shown that enzymatic remodeling of the native antibody glycan at N297 allows the introduction of azide-modified sugars suitable for the attachment of cytotoxic payloads using metal-free click chemistry (see Figure 6). Similarly, enzymatic glycan remodeling protocols can also be used to introduce free thiol groups onto antibodies for conjugation based on any of the methods described above for cysteine ​​conjugation (see Figure 7).

[0013] Although ADCs have demonstrated clinical and preclinical activity, it has been unclear what factors determine such efficacy in addition to antigen expression on targeted tumor cells. For example, drug:antibody ratio (DAR), ADC binding affinity, payload potency, receptor expression level, internalization rate, transport, multidrug resistance (MDR) status, and other factors are all thought to affect the outcome of ADC treatment in vitro. In addition to directly killing antigen-positive tumor cells, ADCs also have the ability to kill adjacent antigen-negative tumor cells: the so-called "bystander killing" effect, as first reported by Sahin et al., Cancer Res. 1990, 50, 6944-6948 (incorporated by reference) and studied, for example, by Li et al., Cancer Res. 2016, 76, 2710-2719 (incorporated by reference). Generally speaking, cytotoxic payloads that are neutral exhibit bystander killing, whereas ionic (charged) payloads do not exhibit bystander killing as a result of the inability of ionic species to readily cross cell membranes by passive diffusion. Payloads with established bystander effects are, for example, MMAE and DXd. An example of a payload that does not exhibit bystander killing is MMAF or the active catabolite (lysine-MCC-DM1) of Kadcyla®.

[0014]

[0014] Currently, cytotoxic payloads include, for example, microtubule-disrupting agents (e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and tubulysin), DNA-damaging agents (e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, duocarmycins, anthracyclines such as PNU-159,682), topoisomerase inhibitors (e.g., DXd, exatecan, and SN-38), or RNA polymerase II inhibitors (e.g., amanitin). ADCs already approved for the market include those with the payloads MMAE, MMAF, DM1, calicheamicin, SN-38, DXd, and PBD dimers. Meanwhile, a BLA has been filed for a DM4-based ADC, and a duocarmycin-based ADC is currently undergoing pivotal trials. A wider variety of payloads are still in clinical evaluation or have been in clinical trials, such as eribulin, indolinobenzodiazepine dimers, PNU-159,682, amanitin, hemiasterlin, doxorubicin, vinca alkaloids, etc. Finally, various ADCs in early clinical or late preclinical stages are conjugated to novel payloads, such as KSP inhibitors, MMADs, cryptophycins, etc.

[0015] With the exception of sacituzumab govitecan (Trodelvy®), all clinical and commercially available ADCs contain cytotoxic drugs that are not suitable as stand-alone drugs. Trodelvy® is an exception because it features SN-38, which is also the active catabolite of irinotecan (the SN-38 prodrug), as its cytotoxic payload. Several other payloads currently used in clinical ADCs were initially evaluated for chemotherapy as free drugs, e.g., calicheamicin, PBD dimer, and eribulin, but have demonstrated extremely high cytotoxic potencies (picomolar to low nanomolar IC ) compared to the typically low micromolar potencies of standard chemotherapeutic agents such as paclitaxel and doxorubicin. 50value).

[0016] Cytotoxins inspired by payloads based on the pyrrolobenzodiazepine (PBD) structure have attracted considerable interest for application in ADCs (see Figure 8). Discovered in the 1960s in cultures of Streptomyces species (e.g., anthramycin and tomaymycin), pyrrolobenzodiazepines are an important class of sequence-selective DNA interactors that covalently bind to guanine bases in the minor groove of DNA. PBD molecules possess a chiral center at their C11a(S)-position, which provides them with the appropriate three-dimensional shape to fit perfectly within the minor groove of DNA. They also possess an electrophilic N10-C11 moiety (i.e., an interconvertible imine, carbinolamine, or carbinolamine methyl ether functional group) that can form a reversible covalent aminal bond between their C11-position and the nucleophilic C2-NH2 of the guanine base. PBD monomers are remarkable in that they possess a three-dimensional shape that allows them to fit perfectly into the DNA groove, due in part to a longitudinal twist caused by the chiral center at their C11a-position. Once positioned in a low-energy position in the groove (i.e., a favorable DNA sequence), determined primarily by the substituents on the A and C rings, the electrophilic C11-position alkylates the C2-NH2 group of the adjacent guanine base, thereby generating a potent covalent adduct that can inhibit biological processes such as transcription factor binding and RNA polymerase progression.

[0017] In addition to PBD monomers, it has been found that chemically covalently linking two PBD monomers together yields PBD dimers with significantly enhanced cytotoxicity (see, e.g., Gregson et al. J. Med. Chem. 2001, 44, 737-748, incorporated by reference). The initial C7 and C8 linkages were designed to span longer DNA lengths than PBD monomers, increasing sequence selectivity and forming DNA crosslinks that may be more difficult for tumor cells to repair. It is now known that PBD dimers can form both interstrand and intrastrand crosslinks and monoadducts under certain conditions, although interstrand crosslink adducts are still considered to be the most toxic in cells. One PBD dimer, SJG-136, was evaluated as a single agent in phase II clinical trials in patients with leukemia or ovarian cancer, but the trials were subsequently discontinued.

[0018] PBD dimers have emerged as ideal candidates for the cytotoxic component of ADCs. The number of potential chemical attachment sites in the PBD provides great flexibility for antibody conjugation, as evidenced by two PBD-drug linker molecules, SGD1910 (taliline) and SG3249 (tesirine), which have been used in numerous ADCs being evaluated in clinical trials. SGD1910 is linked via the C2 position of the PBD, and SG3249 via the N10 position, both via a dipeptide (valine-alanine) trigger that is cleaved by cathepsins in the lysosome. The self-immolative PABC spacer is required to release the bis-imine DNA-bridged PBD dimer from the hemiaminal precursor during ADC catabolism, while the PEG group in SG3249 enhances water solubility. Furthermore, both SGD1910 and SG3249 possess terminal maleimide groups that allow conjugation to antibodies via reaction with cysteine ​​side chain thiols. As reported by Reid et al., ACS Med. Chem. Lett. 2019, 10, 1193-1197 (incorporated by reference), benzoannulated versions of PBDs known as indolinobenzodiazepines (IBDs) are linked to antibodies via an aryl tether in the IBD and are also in clinical development as ADC drug linkers (IMGN779). Again, the linkage in IMGN779 is cleavable (disulfide triggered), but in this case, the payload is attached to the antibody via a lysine linkage. Various other structural analogs of PBD dimers have also been developed over the years, including isoindolinopyrrolobenzodiazepines (IQBs), reported by Smith et al., ACS Med. Chem. Lett. 2018, 9, 56-60 (incorporated by reference). A comprehensive review of PBD dimers was published by Mantaj et al., Angew. Chem. Int. Ed. 2017, 56, 462-488 (incorporated by reference).

[0019] Currently, there is one approved ADC with a PBD dimer payload (Zynlonta™). Zynlonta™ has SG3249 / tesirin as the linker payload, which is also true for at least three other ADCs under development by ADC Therapeutics (ADCT-301, ADCT-602, ADCT-901), and other ADCs such as MT-8633 and TR1801-ADC under development by Tanabe / Medimmune. A derivative of SG3249 / tesirin is also in clinical development by ADC Therapeutics (ADCT-601), which is attached to antibodies via metal-free click conjugation (GlycoConnect™) technology to antibody glycans rather than via maleimide conjugation, as disclosed in Zammarchi et al. Mol. Canc. Ther. 2022, 21, 582-593, which is incorporated by reference. It is noteworthy that numerous PBD-dimer-based ADCs have been introduced into the clinic over the past decade, including ABBV-176, SC16LD6.5 (Rova-T), SC-002, SC-003, SC-004, SC-006, MEDI7247, RG6109 (DCLL9718S), and RG6148 (DHES0815A), but are now discontinued. One reason for the high number of discontinuations is the high potency of PBD-dimers, which can cause serious adverse events in patients, such as edema and exudation, as summarized in J.A. Hartley, Exp. Opin. Biol. Ther. 2020, DOI: 10.1080 / 14712598.2020.1776255, which is incorporated by reference.

[0020] One approach to reducing the toxicity of PBD dimers for ADC applications is to lower the drug load of the antibody; for example, a DAR1 format with the same payload may be preferable because it may have a two-fold higher MTD compared to the analogous DAR2 form. Ruddle et al., ChemMedChem 2019, 14, 1185-1195, recently demonstrated that DAR1 conjugates can be prepared from antibody Fab fragments by introducing a maleimide-based linker attached to Val-Ala-PABC fragments on both PBD monomer fragments of the PBD dimer, thereby generating a symmetric PBD dimer for simultaneous reaction with two reduced interchain cysteines of the Fab (see Figure 10). The resulting DAR1-type Fab fragments were shown to be highly homogeneous, stable in serum, and exhibit excellent cytotoxicity. Subsequent publications, such as White et al., MAbs 2019, 11, 500-515, and further in International Publication No. WO 2019034764 (incorporated by reference), demonstrated that DAR1 conjugates could also be prepared from full IgG antibodies using the same bis-maleimide-functionalized PBD dimer (Figure 10, Flexmab technology). The Flexmab-derived DAR1 ADC was shown to be highly resistant to payload loss in serum and exhibited potent antitumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, this ADC was well tolerated in rats at twice the dose compared to a site-specific DAR2 ADC prepared using a single maleimide-containing PBD dimer.

[0021] To reduce the toxicity and improve the therapeutic window of PBD dimer-based ADCs, next-generation PBD drug-linker design also focuses on using lower-potency PBDs and / or incorporating additional tumor-selective triggers into PBD dimers. For example, after years of neglect, SJG-136, rather than SG3199, the active catabolite released by proteolysis of SG3149, is now being applied to ADCs. In addition to reducing the potency of the DNA-reactive imine that typically remains in ADCs after conjugation, additional capping moieties, such as a β-glucuronidase-cleavable trigger, a peptide-based trigger, or a disulfide-based trigger, can also be incorporated.

[0022] β-Glucuronidase is an enzyme involved in the degradation of endogenous glucuronic acid-containing glycosaminoglycans. It is normally localized in lysosomes in cells and is also present in lysosomes of first-pass tissues such as the liver and intestine. The concentration of β-glucuronidase in many solid tumors, including lung cancer, breast cancer, and gastrointestinal cancer, and in the tumor microenvironment has been reported to be higher than in normal tissues, and this enzyme is not found in the systemic circulation. Furthermore, lysosomal enzymes are released into the extracellular space from dying cells, resulting in elevated levels of these enzymes in necrotic areas of tumors. Lysosomal β-glucuronidase concentrations are also high in inflammatory immune cells, such as neutrophils and eosinophils, which can release the enzyme into inflammatory sites, such as necrotic areas of human tumors. Utilizing the overexpression of β-glucuronidase in certain tumors is a well-known approach in oncology research, and β-glucuronidase-cleavable triggers have been applied to anthracyclines, auristatins, duocarmycins, and camptothecins, as well as multi-compound release prodrug approaches, PBD prodrugs, and ADCs. For example, Gregson et al., Eur. J. Med. Chem. 2019, 179, 591-607, discloses an asymmetric PBD dimer (conjugated via maleimide) linked to an antibody via a Val-Ala-PABC element, with a β-glucuronidase-cleavable trigger element on the other PBD monomer (Figure 11). Similar masked PBD dimers based on glucuronic acid have also been disclosed, for example, in U.S. Patent No. 20220218830 and International Publication No. 2020141923.

[0023]

[0023] Other enzymes upregulated in the tumor microenvironment, including cathepsins, matrix metalloproteinases (MMPs), legumain, and the serine protease elastase, have also been considered for the release of cytotoxic payloads, as summarized in, for example, M. Poreba, FEBS J., 2020, 287, 1936-1969. Investigations of these two TME-associated enzymes have led to the development of cleavable linkers based on specific peptide sequences, for example, Val-Cit or Val-Ala for cathepsins, Pro-Leu-Gly (PLG) for metalloproteinases (MMPs), Asn-Asn, Ala-Asn, or Asn-Ala for legumain, and Asn-Pro-Val (NPV) for the serine protease elastase.

[0024]

[0024] Finally, it is also known in the art that disulfide-based linkers can be used as selective triggers to release active catabolic products from ADCs containing PBD dimer payloads (see Figure 12), taking advantage of the fact that the cytoplasm is significantly more reducing than the extracellular environment.

[0025] [Summary of the Invention] The present inventors have surprisingly discovered that conjugating a masked PBD dimer via the glycan of a cell-binding agent increases the efficacy and / or tolerability of the PBD-payload. These findings may allow for more effective cancer treatments with fewer adverse side effects.

[0026] The present invention relates first to an antibody drug conjugate having the structure (1): AB-[L-(D) x ] y (1) (In the ceremony AB is a cell binding agent; x is 1 or 2; y is 1 or 2; D is the masked PBD dimer payload; L is a linker connecting AB to D, and L is at least one -L 6 -Z-fragment, Z 1 is a linking group containing the product of the cycloaddition reaction, and L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - and L 6 is GlcNAc(Fuc) w is connected to AB via: G is a monosaccharide, j is an integer ranging from 0 to 6, S is a sugar or sugar derivative; GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1; w' is 0 or 1, L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-).

[0027]

[0027] The present invention further relates to a process for the synthesis of an antibody-drug conjugate according to the present invention, a linker-drug construct suitable for use in the process according to the present invention, medical uses of the antibody-drug conjugate according to the present invention, and a pharmaceutical composition comprising the antibody-drug conjugate according to the present invention. [Brief explanation of the drawings]

[0028] [Figure 1] A general scheme for the preparation of antibody-drug conjugates by reaction of a monoclonal antibody (most often a symmetric dimer) containing x functional groups F is shown. By incubating antibody-(F)x with an excess of linker-drug construct (Q-spacer-linker-payload), a conjugate is obtained by reaction of F with Q to form a linking group Z. [Figure 2]Various reagents suitable for reaction with cysteine ​​side chains are shown. The reagents can be of the mono-alkylating type (A) or cross-linking agents (B) for reaction with two cysteine ​​side chains. [Figure 3] Figure 1 shows a general process for the non-genetic conversion of a monoclonal antibody (mAb) into an antibody containing probes for click conjugation (F). The click probes can be located at various positions in the antibody depending on the technique used. For example, an antibody can be converted into an antibody containing two click probes (structure on the left), four click probes (structure on the bottom), or eight probes (structure on the right) for click conjugation. [Figure 4] Figure 4 shows a representative (but not comprehensive) set of functional groups (F) that can be introduced into antibodies by genetic engineering, chemical modification, or enzymatic means, which, upon metal-free click reaction with a complementary reactive group Q, yield a linking group Z. The functional group F can be artificially introduced (engineered) into antibodies at any optimal position. Some functional groups F (e.g., nitrile oxides, quinones) can also react with strained alkenes in addition to strained alkynes, as shown for triazines or tetrazines (bottom row) as examples. The pyridine or pyridazine linking group is the product of rearrangement of the tetrazabicyclo[2.2.2]octane linking group, with loss of N, formed upon reaction of triazines or tetrazines with alkynes (but not alkenes), respectively. The linking group Z shown in Figure 4 is a preferred linking group for use in the present invention. [Figure 5] Preferred embodiments are given for cyclic alkynes suitable for metal-free click chemistry, and reactive moieties Q. This list is not exhaustive, for example, alkynes can be further activated by fluorination, aromatic ring substitution, or introduction of heteroatoms in the aromatic ring. [Figure 6]We demonstrate the glycan remodeling of a full-length IgG followed by site-specific conjugation of a payload based on azido-cyclooctyne click chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all distinct glycoforms, followed by glycosyltransferase-mediated transfer of the azido sugar onto the core GlcNAc, which is released by the endoglycosidase. In the next step, the azido-remodeled IgG is subjected to a single cyclooctyne-modified immune cell-engaging polypeptide for metal-free click chemistry (SPAAC), resulting in a bispecific antibody in a 2:2 molecular format. We also demonstrate that the cyclooctyne-polypeptide construct has a specific spacer between the cyclooctyne and the polypeptide, which allows for tuning of the IgG polypeptide distance or imparts other properties to the resulting bispecific antibody. [Figure 7] We demonstrate a specific example of glycan remodeling of full-length IgG followed by site-specific conjugation of a payload based on thiol alkylation chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of thiol-modified (and disulfide-protected) sugar derivatives onto the core GlcNAc released by the endoglycosidase. In the next step, the remodeled IgG is subjected to reduction (to convert disulfides to thiols), optionally followed by oxidation, and then reaction with a payload modified with an appropriate thiol-reactive reagent. [Figure 8] The general structure of a pyrrolobenzodiazepine (PBD) monomer (with carbon numbering) and a typical structure of a PBD dimer are shown, as well as the specific chemical structures of tomaymycin, anthramycin, and SJG-136. [Figure 9] 1 shows the structures of SGD1910 (Taliline), SG3149 (Tesiline), which are suitable for cysteine ​​conjugation to proteins, and IMGN779, which is suitable for lysine conjugation to proteins (e.g., antibodies). [Figure 10] Figure 1 shows the structure of a symmetrical bismaleimide-functionalized PBD dimer SG3199 via two Val-Ala-PABC cleavable elements for dual conjugation of proteins with (at least) two freely available cysteine ​​side chains. [Figure 11] We show the structure of an asymmetrically modified PBD dimer, modified on one side via a consensus Val-Ala-PABC element that is released by endogenous proteases (e.g., cathepsin B) and with a glucuronic acid-functionalized p-hydroxybenzyloxycarbonyl group on the other side that is released by b-glucuronidase. [Figure 12] We demonstrate how a disulfide-based cleavable element can be introduced to link a PBD dimer to an antibody cysteine ​​(e.g., with glutathione) for reductive release of an active catabolic product. [Figure 13] Figure 1 shows the in vivo efficacy in a mouse xenograft model (JIMT-1 cell line-derived xenograft model, HER2 1+). The ADCs evaluated were trast-E (1 mg / kg and 3 mg / kg) and B12-E (3 mg / kg). The tumor volume of the mice was monitored (top graph). Data are plotted as mean + / - standard error. [Figure 14] This figure shows the time-dependent changes in body weight of SD rats administered a single bolus of vehicle (PBS pH 7.4) or trast-E (10, 20, 30 mg / kg) on ​​day 0 and then observed for 3 weeks. The rats' body weights were monitored (top graph). The percentage of body weight change was calculated relative to day 0 (bottom graph).

[0029] [Detailed Description of the Invention] [Definition]

[0042] As used in this specification and claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0030]

[0043] The compounds disclosed herein and in the claims may contain one or more asymmetric centers, and the compounds may exist in different diastereomers and / or enantiomers. Reference to any compound in this specification and claims is intended to include all diastereomers and mixtures thereof, unless otherwise specified. Furthermore, reference to any compound in this specification and claims is intended to include both individual enantiomers and any mixtures of enantiomers, racemates, etc., unless otherwise specified. When the structure of a compound is depicted as a specific enantiomer, it should be understood that the invention of this application is not limited to that specific enantiomer.

[0031]

[0044] Compounds can exist in different tautomeric forms. The compounds according to the present invention are intended to include all tautomeric forms unless otherwise specified. When the structure of a compound is shown as a specific tautomer, it should be understood that the invention of this application is not limited to that specific tautomer.

[0032]

[0045] The compounds disclosed herein and in the claims can further exist as R and S stereoisomers. Unless otherwise specified, the description of any compound in the specification and claims is intended to include both the individual R and individual S stereoisomers of the compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific S or R stereoisomer, it should be understood that the invention of this application is not limited to that specific S or R stereoisomer.

[0033]

[0046] The compounds disclosed herein and in the claims may further exist as exo and endo diastereoisomers. Unless otherwise specified, the description of any compound in the specification and claims is intended to include both the individual exo and individual endo diastereomers of the compound, as well as mixtures thereof. When the structure of a compound is depicted as a particular endo or exo diastereomer, it should be understood that the invention of this application is not limited to that particular endo or exo diastereomer.

[0034]

[0047] The compound according to the present invention may exist in the form of a salt, and the salt form is also included in the present invention.The salt is typically a pharmaceutically acceptable salt containing a pharmaceutically acceptable anion.The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced with a cation such as a metal cation or an organic cation.If applicable, the salt is a pharmaceutically acceptable salt, but this is not required for salts that are not intended for administration to patients.For example, in the salt of a compound, the compound can be protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0035]

[0048] The term "pharmaceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that is mammalian safe for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which are derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and, when the molecule contains a basic functional group, organic or inorganic acid salts such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0036]

[0049] The term "protein" is used herein in its ordinary scientific sense, where a polypeptide containing about 100 or more amino acids is considered a protein. Proteins contain natural amino acids, but may also contain unnatural amino acids.

[0037]

[0050] The term "cell-binding agent" is used herein to define an agent, typically a chemical moiety or (poly)peptide, that specifically binds to a cell. Typically, a cell-binding agent binds to an epitope. Cell-binding agents include antibodies, B cells, and T cells.

[0038]

[0051] The term "antibody" is used herein in its ordinary scientific sense. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" includes whole immunoglobulins, but is also intended to include antigen-binding fragments of antibodies. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0039]

[0052] An "antibody fragment" is defined herein as a portion of an intact antibody comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, minibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0040]

[0053] An "antigen" is defined as the entity to which an antibody specifically binds.

[0041]

[0054] The terms "specific binding" and "specifically bind" are defined herein as a highly selective manner in which an antibody selectively binds to a corresponding epitope of its target antigen and not to many other antigens. Typically, an antibody or antibody derivative binds with an affinity of at least about 1 x 10 M, preferably 10 M to 10 M, 10 M, 10 M, or 10 M, and binds to a given antigen with an affinity that is at least twice its binding affinity to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0042]

[0055] The terms "substantial" or "substantially" are defined herein as the majority of the population of a mixture or sample, i.e., >50%, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0043]

[0056] The term "glycan" is used herein in its ordinary scientific sense to refer to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term glycan refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of one sugar, which may be unsubstituted (monosaccharide) or may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when a longer sugar chain is attached to a protein, the sugar chain is also considered a glycan herein. The glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man), or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to a protein and is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but is attached to a single other sugar, i.e., a sugar that does not have an additional sugar substituent on one or more of its other hydroxyl groups, is called a terminal sugar. For the avoidance of doubt, an oligosaccharide of a glycoprotein may have multiple terminal sugars, but only one core sugar. Glycans can be O-linked, N-linked, or C-linked glycans. In O-linked glycans, monosaccharides or oligosaccharide glycans are attached to an O-atom in an amino acid of the protein, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, monosaccharides or oligosaccharide glycans are attached to a protein via an N atom in an amino acid of the protein, typically via the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In C-linked glycans, a monosaccharide or oligosaccharide glycan is attached to a C atom in an amino acid of a protein, typically the C atom of tryptophan (Trp).

[0044]

[0057] Linker is defined herein as the part that connects two or more elements of a compound.For example, in antibody conjugate, antibody and payload are covalently linked to each other through linker.Linker can comprise one or more linkers and spacer part that connects various parts in linker.

[0045]

[0058] A "spacer" or spacer moiety is defined herein as a moiety that covalently attaches two (or more) parts of a linker in a spaced-apart manner (i.e., providing distance between them). A linker may be, for example, part of a linker construct, linker conjugate, or bioconjugate, e.g., as defined below.

[0046]

[0059] As used herein, a "self-immolative group" is defined as a portion of the linker of an antibody-drug conjugate that functions to conditionally release a free drug at a site targeted by a ligand unit. The activatable self-immolative moiety comprises an activatable group (AG) and a self-immolative spacer unit. When the activatable group is activated, for example, by enzymatic conversion of an amide group to an amino group or by reduction of a disulfide to a free thiol group, a self-immolative reaction sequence is initiated, which results in the release of the free drug by one or more of a variety of mechanisms. This includes (transient) 1,6-elimination of a p-aminobenzyl group to a p-quinone methide or 1,6-elimination of a p-hydroxybenzyl group to a p-quinone, optionally with the release of carbon dioxide and / or a secondary cyclization release mechanism. The self-immolative assembly unit can be part of a chemical spacer (via a functional group) that links the antibody and payload. Alternatively, the self-immolative group is not an inherent part of the chemical spacer, but is branched from the chemical spacer that links the antibody and payload.

[0047]

[0060] A "conjugate" is defined herein as a compound in which a cell-binding agent is covalently attached to a payload via a linker. A conjugate comprises one or more cell-binding agents and / or one or more payloads.

[0048]

[0061] The term "payload" refers to a moiety that is covalently attached to a targeting moiety, such as an antibody, but also to a molecule that is released from the conjugate upon uptake of the protein conjugate and / or cleavage of the linker. Thus, payload refers to a monovalent moiety with one open end that is covalently attached to a targeting moiety via a linker, and also to a molecule that is released therefrom. In the context of the present invention, the payload is a masked PBD dimer.

[0049]

[0062] A "hydrophilic group," "hydrophilic moiety," or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that confer improved polarity and therefore improved aqueous solubility to the molecule to which it is attached. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonate groups, phosphate groups, acylsulfamide groups, or carbamoylsulfamide groups. The hydrophilic moiety can be selected from sulfonated side chains, carbamoylsulfamide side chains, polysarcosine-containing side chains, or polyethylene glycol-containing side chains. More preferably, the hydrophilic moiety is selected from a carbamoylsulfamide side chain or a polyethylene glycol side chain, and most preferably, the hydrophilic moiety is selected from a polyethylene glycol side chain. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency, less aggregation, and improved pharmacokinetics when incorporated into antibody-drug conjugates, thereby resulting in higher efficacy and in vivo tolerability.

[0050]

[0063] "PBD dimers" or "pyrrolobenzodiazepine dimers" are a class of cytotoxic agents used in cancer therapy, particularly as payloads for antibody-drug conjugates. PBDs possess a chiral center at the C11a(S)-position (see Figure 8), providing them with a suitable three-dimensional shape that fits perfectly within the DNA groove. "Masked PBD dimers" are defined as pyrrolobenzodiazepine dimer derivatives containing at least one cap, where the cap is hydrophilic and contains a self-immolative group. The purpose of the cap is to mask the imine group present in the active catabolite as a hemiaminal group, reducing the toxicity of the PBD until the payload reaches its target. The cap is removed by conditions specific to the tumor microenvironment or intracellular environment, which results in the release of the active PBD catabolite via the initial release of the self-immolative group. Such capped PBD dimers can be considered as prodrugs of the PBD dimer. A masked PBD dimer contains at least one cap that is linked to a hemiaminal or amine position at one end of the PBD dimer, but can also be linked to a hemiaminal or amine position at the other end of the PBD dimer when the PBD dimer is conjugated to an antibody via a spacer linking the two PBD monomers. Thus, in a masked PBD dimer, both imine groups are typically protected by either a linker or a cap, and the imine groups are deprotected in the tumor microenvironment and / or intracellular environment.

[0051]

[0064] The number of payload (D) molecules conjugated to a single cell-binding agent, e.g., an antibody, is known in the art as the DAR (drug-antibody ratio). These are theoretical DAR values; it will be understood that in practice, the DAR may deviate slightly from this value. Typically, conjugates are obtained as stochastic mixtures of antibody-drug conjugates, and DAR values ​​vary among individual conjugates. Depending on the conjugation technique used, the DAR may have a wide distribution (e.g., DAR = 0-10) or a narrow distribution (e.g., DAR = 3-4). For such mixtures, the DAR often refers to the average DAR of the mixture. This is well known in the bioconjugation art. However, when conjugation occurs via glycans, as in the present invention, antibody-drug conjugates have DARs close to the theoretical value. For example, if the theoretical DAR is 4, AR values ​​greater than 3.6 or even greater than 3.8 are readily obtained, indicating that most antibodies in the reaction mixture are fully reacted and have a DAR of 4. Furthermore, DAR values ​​greater than 4.0 are not observed.

[0052]

[0065] "DAR# conjugate" refers to an antibody-drug conjugate, where DAR# indicates the number of payloads per antibody. In other words, a DAR1 conjugate is a conjugate containing one payload, a DAR2 is a conjugate containing two payloads, and a DAR4 conjugate is a conjugate containing four payloads. In the context of the present invention, a DAR4 conjugate contains four masked PBD payloads, and thus four pyrrolobenzodiazepine dimers.

[0053]

[0066] The term "bystander killing" or "bystander effect" refers to cell death of neighboring cells due to passive diffusion of a drug from a target cell to the neighboring cells.

[0054] [The present invention]

[0067] The present inventors have developed antibody-drug conjugates containing a masked PBD as a cytotoxic payload, which have no or negligible tendency to aggregate. The ACDs according to the present invention were found to exhibit significant in vivo efficacy. Furthermore, the therapeutic index of the antibody-conjugates according to the present invention was increased compared to conventional PBD dimer-based antibody-drug conjugates, demonstrating the beneficial effect of applying a masked PBD via the glycan of a cell-binding agent based on metal-free click chemistry and linking the linker-drug.

[0055]

[0068] The present invention relates first to antibody-drug conjugates of general structure (1): AB-[L-(D) x ] y (1)

[0056]

[0069] In a second aspect, the present invention relates to a process for the synthesis of an antibody-drug conjugate according to the invention. In a third aspect, the present invention relates to a linker-drug construct suitable for use in the process according to the invention. In a fourth aspect, the present invention relates to medical uses of an antibody-drug conjugate according to the invention, and to pharmaceutical compositions comprising an antibody-drug conjugate according to the invention. Those skilled in the art will understand that all aspects are related, and that everything said about an antibody-drug conjugate according to the invention applies equally to the process according to the invention, the linker-drug construct according to the invention, the use according to the invention, and the composition according to the invention, and vice versa.

[0057] [Antibody-drug conjugates]

[0070] The antibody-drug conjugate according to the present invention has the following structure (1): AB-[L-(D) x ] y (1) (In the ceremony AB is a cell binding agent; x is 1 or 2; y is 1 or 2; D is the masked PBD payload. L is a linker connecting AB to D, and L is at least one -L 6 -Z 1 - contains fragments, Z 1 is a linking group, said linking group comprising the product of a cycloaddition reaction; L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - and L 6 is GlcNAc(Fuc) w is connected to AB via: G is a monosaccharide, j is an integer ranging from 0 to 6, S is a sugar or sugar derivative; GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1; w' is 0 or 1, L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-).

[0058]

[0071] AB represents a cell binding agent. Preferably, the cell binding agent is an antibody. Those skilled in the art will understand that the present invention can be applied to any antibody. More preferably, the conjugate according to the present invention comprises an antibody that interacts with a receptor overexpressed in cell carcinoma.

[0059]

[0072] The antibody-drug conjugate may be a DAR1, DAR2, or DAR4 conjugate. In the case of a DAR1 conjugate, x and y in structure (1) are both 1. A DAR2 conjugate is represented by structure (1), where x=1 and y=2, or x=2 and y=1, preferably x=1 and y=2. A DAR4 conjugate is represented by structure (1), where x is 2 and y is also 2. Preferably, the antibody-drug conjugate is a DAR1 conjugate or a DAR2 conjugate, and more preferably, the antibody-drug conjugate is a DAR2 conjugate.

[0060]

[0073] L is a linker that connects the antibody to the payload, D. The linker L is selected from at least one -L 6 -Z 1 -Fragment, L 6 is linked to the antibody. The structure of the linker is determined by whether the conjugate is a DAR1, DAR2, or DAR4 conjugate. When the conjugate is a DAR1 conjugate with y=1 and x=1, L preferably conforms to structure (2): [ka] (wherein both L 6 is concatenated into a single AB, and L M is connected to D, and (Z 2 ) z’ represents an optional Z-fragment which can be present (z'=1) or absent (z'=0).

[0061]

[0074] For DAR2 and DAR4 conjugates where y=2, L is preferably represented by structure (3): [ka] (In the formula, L 6 is connected to AB, and L M is connected to D).

[0062] [Mode of conjugation - Linker L 6 ]

[0075] The conjugate according to the invention is Click-Probe F 1 The modified antibody containing a sugar or sugar derivative having a payload and a click-probe Q 1 It can be prepared by reacting with a compound comprising

[0063]

[0076] The mode of conjugation includes conjugation via the glycans of the antibody, preferably via N-glycosylation sites. 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, where j is an integer ranging from 0 to 6, w is 0 or 1, w' is 0 or 1, GlcNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, G is a monosaccharide moiety, and S is a sugar or sugar derivative. 6 teeth -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, where GlcNAc(Fuc) w - is attached to the peptide portion of the antibody, and S is Z 1 or F 1 where GlcNAc is a core N-acetylglucosamine moiety typically present in the glycan structure of an antibody. Here, the core N-acetylglucosamine moiety refers to an N-acetylglucosamine moiety directly attached to the peptide chain of an antibody. This core N-acetylglucosamine moiety is optionally fucosylated (d is 0 or 1), which is a general characteristic of antibodies.

[0064]

[0077] (G) jrepresents the glycoform of the antibody. The present invention can be applied to antibodies of any glycoform. Typical monosaccharides present in glycans from which G can be selected include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and xylose. Thus, (G) j can be a linear or branched oligosaccharide containing j monosaccharide moieties. Typical glycans have j ranging from 4 to 16, preferably 6 to 10. In a preferred embodiment, the antibody is trimmed and j=0. Such trimming can be performed by an endoglycosidase enzyme such as EndoS. Glycan-mediated conjugation preferably uses an N-glycosylation site, more preferably an N-glycosylation site linked to an asparagine amino acid of the antibody, most preferably the conserved glycosylation site at amino acid N297 of the antibody. Typically, L 6 is formed at least in part by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at an asparagine residue at or near position 297 (Kabat numbering) of the heavy chain, which is decorated with complex-type glycans. This naturally occurring glycosylation site of the antibody is preferably used, although other glycosylation sites, including artificially introduced ones, may also be used in the linker L. 6 Thus, in a preferred embodiment, L 6 is linked to an amino acid of the antibody located at a position in the range of 250 to 350 of the heavy chain, preferably at a position in the range of 280 to 310 of the heavy chain, more preferably at a position in the range of 295 to 300 of the heavy chain, and most preferably at position 297 of the heavy chain.

[0065]

[0078] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan or a part thereof. Thus, the -GlcNAc(Fuc) of the glycan w -(G) j- is typically derived from the original antibody, GlcNAc is an N-acetylglucosamine moiety, and Fuc is a fucose moiety. Fuc is typically attached to GlcNAc via a β-1,6-glycosidic bond. Usually, antibodies can be fucosylated (w=1) or non-fucosylated (w=0). In the context of the present invention, the presence of the fucosyl moiety is irrelevant, and fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates achieve similar results. The GlcNAc residue, sometimes referred to as the core-GlcNAc residue, is the monosaccharide that is directly attached to the peptide portion of the antibody.

[0066]

[0079] S is core-GlcNAc(Fuc) w The moiety can be directly linked to the core-GlcNAc(Fuc) moiety, i.e., j=0, which means that the remainder of the glycan is core-GlcNAc(Fuc) before S is attached. w Such glycan trimming is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, the core-GlcNAc(Fuc) w There are one or more monosaccharide residues between the moiety and S, where j is typically an integer ranging from 1 to 10, preferably j=2 to 5. In a preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer ranging from 2 to 5. (G) j is typically linked to GlcNAc(Fuc) via a β-1,4-linkage wIn a preferred embodiment, j is 3, 4, or 5. While any monosaccharide that may be present in a glycan 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. Antibodies and antibody conjugates with j = 0 exhibit no or significantly reduced binding to Fc-γ receptors, while antibodies and antibody conjugates with j in the range of 4 to 10 bind to Fc-γ receptors. Therefore, by selecting a specific value for j, the desired degree of binding to Fc-γ receptors can be obtained. Therefore, j = 0, 4, 5, 6, 7, 8, 9, or 10 is preferred, more preferably j = 0, 4, or 5, and most preferably the antibody is trimmed and j = 0.

[0067]

[0080] S is a sugar or sugar derivative. The term "sugar derivative" is used herein to refer to a monosaccharide sugar, i.e., a derivative of a monosaccharide containing a substituent and / or functional group. Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH), galactosamine (GalNH), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (SialA), also known as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). In the present invention, S is linked to a linking group Z after conjugation with a cell-binding moiety. 1 Click probe F converted to 1 Preferably, S is selected from Gal, GlcNAc, GalNAc, and NeuNAc. In a particularly preferred embodiment, S is GalNAc. Most preferably, S is a monosaccharide modified to contain F at position 2 or 6. 1 modified with (or Z 1(after conjugation with) N-acetylgalactosamine.

[0068]

[0081] Linking group Z 1 or reactive group F 1 may be directly bonded to S, or S and Z 1 Or F 1 Between them is the linker L 7 may be present. Therefore, L 7 may be present (w'=1 or 2) or absent (w'=0). Typically, each moiety Z 1 is the linker L 7 and thus in one embodiment, x with w'=0. Preferably, L 7 does not exist, and each connecting part Z 1 is directly bonded to S. If present, L 7 teeth It may be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. In a preferred embodiment, x=1 and w'=0 or 1, and most preferably, x=1 and w'=0.

[0069]

[0082] y represents x reactive groups F 1 or x number of bonding groups Z connected to AB 1 where y is an integer indicating the number of sugar (derivative) S linked to x. y is 1 or 2, preferably y=2. Thus, an antibody contains y moieties S, each of which contains x reactive groups F. These reactive groups F 1 is reacted with a reactive moiety Q of the linker-toxin construct, thereby forming an x×y linking group Z 1 is formed, and x×y payloads are attached to a single AB. Each linker-toxin construct can contain multiple payloads, for example, by a branching moiety BM in L. Preferably, each linker-toxin construct contains one or two occurrences of D, most preferably one occurrence of D. In a particularly preferred embodiment, the linker L 1 contains a branching moiety to which the second occurrence of D is linked.

[0070]

[0083] x is a linking group Z bonded to the sugar (derivative) S 1 or reactive group F 1 is an integer indicating the number of reactive moieties F. Thus, an antibody preferably has x reactive moieties F 1 These reactive moieties F containing moieties S containing 1 each of which is a reactive moiety Q of the linker-toxin construct 1 thereby reacting with x number of linking groups Z 1 is formed, and x payloads are combined into a single occurrence of S, where x is 1 or 2, preferably x=1.

[0071] [Linking group Z]

[0084] Z is a linking group. The term "linking group" refers to a structural element that connects one part of a conjugate to another part of the same bioconjugate. In the present invention, the antibody AB is linked to the masked PBD payload via a linker. The linking group Z 1 is a moiety obtainable by cycloaddition, preferably a metal-free click reaction, and the reactant of this cycloaddition is a click probe F 1 and Click Probe Q 1 Those skilled in the art will recognize that Z 1 The true nature of F 1 and Q 1 Understand that it depends on the nature of 1 and F 1 Preferred embodiments of are defined below.

[0072]

[0085] Z 1 is formed by cycloaddition. Conjugation reactions via cycloaddition are known to those skilled in the art, and the skilled artisan will be able to identify the appropriate reaction partner F 1 and Q 1 can be selected, and the resulting linking group Z 1The nature of the conjugation will be understood. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reaction) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably Q 1 is or contains an alkyne group, F 1 is an azido group. Cycloadditions such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art and one of ordinary skill in the art would know how to perform them.

[0073]

[0086] Preferably, Z 1 contains a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide, or imide group. The triazole moiety is 1 In one embodiment, Z 1 contains a (hetero)cycloalkene moiety, i.e., Q contains a (hetero)cycloalkyne moiety 1 In an alternative embodiment, Z 1 contains a (hetero)cycloalkane moiety, i.e., Q contains a (hetero)cycloalkene moiety 1 As used herein, aromatic rings such as triazole rings are considered heterocycloalkane rings because they are formed by the reaction of an alkyne moiety with an azide moiety. In a preferred embodiment, Z 1 has the structure (Z1): [ka]

[0074]

[0087] During the ceremony, [ka] A bond shown as is a single or double bond. Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za) to (Zj) as defined below, wherein: ** The carbon atom labeled with is the carbon atom of (Z1) to which ring Z is fused. [ka] corresponding to the two carbon atoms of the bond shown as: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31and S (+) is B (-) is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked 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 = an integer in the range of 8 to 16; Ring Z is formed by cycloaddition and is preferably selected from (Za) to (Zj).

[0075]

[0088] In a preferred embodiment, u+u′=0, 4, 5, 6, 7 or 8, more preferably 0, 4 or 5. [ka] When the bond shown as is a double bond, it is preferred that u+u'=4, 5, 6, 7 or 8, more preferably u+u'=4 or 5. [ka] When the bond shown as is a single bond, it is preferred that u+u'=0 or 5. * The wavy bonds labeled with are sometimes L 6 is connected to AB via ** The wavy bond labeled with is connected to L.

[0076]

[0089] Z 1 It is particularly preferred that the formula (I) contains a (hetero)cycloalkene moiety, i.e. [ka] In a preferred embodiment, the bond shown as Z 1is selected from structures (Z2) to (Z20) or (Z38a): [ka]

[0077]

[0090] where the link to the payload via the linker is represented by a wavy bond, and B (-) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by a cycloaddition reaction and is preferably selected from triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za) to (Zj) shown below, where: ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z2) to (Z20) and (Z38a) to which the ring Z is fused. In the context of this embodiment, the linking group Z is formed by reaction with a (hetero)cycloalkyne, and therefore, [ka] A bond shown as is a double bond. [ka]

[0078]

[0091] In a further preferred embodiment, Z 1 is selected from structures (Z21) to (Z38a) shown below: [ka]

[0079]

[0092] wherein the connection to L is represented by a wavy bond. Structure (Z29) can be in the endo or exo configuration, preferably in the endo configuration. In structure (Z38), B(-) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) to (Zj) defined above.

[0080]

[0100] In a preferred embodiment, Z 1 preferably comprises a (hetero)cyclooctene or (hetero)cycloheptene moiety according to the structures (Z8), (Z26), (Z27), (Z28) or (Z37), which are optionally substituted. Z 1 Each of these preferred options for is further defined herein below.

[0081]

[0093] Therefore, in a preferred embodiment, Z 1 comprises an optionally substituted heterocycloheptene moiety according to structure (Z37). Preferably, the heterocycloheptene moiety according to structure (Z37) is unsubstituted.

[0082]

[0094] In a preferred embodiment, Z 1 comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z 1 preferably comprises a (hetero)cyclooctene moiety according to the structure (Z39) shown below, where V is (CH) l where l is an integer ranging from 0 to 10, preferably from 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably, l is 0, 1 or 2, and most preferably, l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z 1 conforms to structure (Z42), further defined below.

[0083]

[0095] In an alternative preferred embodiment, Z 1comprises a (hetero)cyclooctene moiety according to the structure (Z26), (Z27) or (Z28), which are optionally substituted. In the context of this embodiment, Z 1 preferably comprises a (hetero)cyclooctene moiety according to the structure (Z40) or (Z41) shown below, where Y 1 is O or NR 11 and R 11 are independently hydrogen, straight or branched chain C1-C 12 Alkyl group or C4-C 12 (Hetero)aryl groups. The aromatic ring of (Z40) is optionally O-sulfonylated at one or more positions, while the ring of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety of structure (Z40) or (Z41) is not further substituted. Most preferably, Z 1 conforms to structure (Z43), further defined below.

[0084]

[0096] In an alternative preferred embodiment, Z 1 contains a heterocycloheptenyl group and conforms to structure (Z37). [ka]

[0085]

[0097] In a particularly preferred embodiment, Z 1 contains a cyclooctenyl group and conforms to structure (Z42): [ka]

[0086]

[0098] During the ceremony: * The bond labeled with is linked to AB, ** The wavy bond labeled with is linked to L;

[0099] R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups;

[0100] R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups;

[0101] R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is linked via a spacer moiety to Z 1(or Q) or the second occurrence of D; and l is an integer ranging from 0 to 10.

[0087]

[0102] In a preferred embodiment of the group according to structure (Z42), R 15 is hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 15 is hydrogen or C1-C6 alkyl, more preferably R 16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the group according to structure (Z42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the group according to structure (Z42), R 19 is H. In preferred embodiments of groups according to structure (Z42), i is 0 or 1, and more preferably 1 is 1.

[0088]

[0103] In a particularly preferred embodiment, Z 1 contains a (hetero)cyclooctenyl group and conforms to structure (Z43): [ka]

[0089]

[0104] where: the bond labeled * is connected to AB, the wavy bond labeled ** is connected to L; R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24and (hetero)arylalkyl groups, wherein the alkyl group, the (hetero)aryl group and the (hetero)arylalkyl group are optionally substituted and include two substituents R 15 are joined together to form an optionally substituted cyclized cycloalkyl or optionally substituted cyclized (hetero)arene substituent R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; Y is independently selected from the group consisting of N or CR 15 is.

[0090]

[0105] In a preferred embodiment of the group according to structure (Z43), R 15 is hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 is hydrogen and -S(O)3 (-) In preferred embodiments of groups according to structure (Z43), Y is N or CH, more preferably Y=N.

[0091]

[0106] In a particularly preferred embodiment, Z 1 contains a heterocycloheptenyl group according to structure (Z37) or (Z38a), and ring Z is a triazole. [ka]

[0092]

[0107] In an alternative preferred embodiment, Z 1 represents a (hetero)cycloalkane moiety, i.e., [ka] The bond shown above as is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkyl group or a cycloalkyl group, preferably a cycloalkyl group, where 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 a (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, where the (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group is optionally substituted. Preferably, Z 1 includes a cyclopropyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornane or norbornene group according to structure (Z46), a (hetero)cycloheptyl moiety according to structure (Z47), or a (hetero)cyclooctyl moiety according to structure (Z48). 3 is C(R 23 )2, NR 23 or O, and each R 23 are individually hydrogen, C1-C6 alkyl, or optionally bonded to L via a spacer and labeled [ka] is a single or double bond. In a further preferred embodiment, the cyclopropyl group conforms to structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group conforms to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group conforms to structure (Z52), (Z53), (Z54), (Z55), or (Z56). [ka]

[0093]

[0108] wherein the R group on Si in (Z50) and (Z51) is typically alkyl or aryl, preferably C1-C6 alkyl; Ring Z is selected from structures (Zk) to (Zn); ** The carbon atoms labeled with correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z44) to (Z56) to which ring Z is fused, * The carbon labeled with is linked to AB. 1 is formed in the context of this embodiment by reaction with a (hetero)cycloalkene, [ka] The bond shown above as is a single bond. [ka]

[0094]

[0109] Z 2 is a further linking group that may typically be present in the DAR1 conjugate when y=1. 2 is formed by reacting a precursor containing the payload with a precursor containing the other portion of the linker. 2 The nature of the linking group Z may take any form, depending on the nature of the reactive moieties and the type of reaction carried out to link these moieties. 2 is selected from the group consisting of an amide moiety, an ester moiety, a carbamate moiety, a carbonate moiety or a (hetero)aryl moiety, a (hetero)cycloalkene moiety, a cycloalkane moiety, more preferably an amide moiety or a carbamate moiety. 2 can be formed by a nucleophilic reaction, preferably a nucleophilic substitution reaction or a Michael addition reaction. In a particularly preferred embodiment, Z 2 is formed by a click reaction, in this case Z 2 contains a (hetero)aryl moiety, a (hetero)cycloalkene moiety or a heterocycloalkane moiety, and most preferably Z 2 Z, including its preferred embodiments 1In this embodiment, Z 1 and Z 2 may be individually selected, and Z 1 It is preferred that both occurrences of are identical.

[0095] [Branch part]

[0110] In a preferred embodiment, the linker of the conjugate according to the present invention contains a branched moiety. A "branched moiety" in the context of the present invention refers to a moiety embedded in a linker that connects three moieties. In other words, a branched moiety contains at least three bonds to other moieties. In one embodiment, the conjugate is a DAR4 conjugate, in which the BM contains one bond to antibody AB, one bond to a PBD dimer payload, and one bond to a second PBD dimer payload. Alternatively, the conjugate is a DAR1 conjugate, in which the BM contains one bond to a PBD dimer payload, one bond to antibody AB, and another bond to the same antibody AB. Any moiety containing at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. In a preferred embodiment, the branched moiety BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring, or a polycyclic moiety. Most preferably, the branched moiety is a nitrogen atom.

[0096] [Linker L B and L C ]

[0111] L B is a chain of at least 2, preferably 5 to 100 atoms selected from C, N, O, S and P, which may typically be present in the DAR1 conjugate when y=1, and the branched portion of the moiety BM is L 6 Z adjacent to 1 Connect to L B is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 The alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may optionally be substituted, and the groups may optionally be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ and NR 21 wherein y' is 0, 1 or 2, preferably y'=2, and R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups.

[0097]

[0112] In a preferred embodiment, L B Both occurrences of are identical. Preferred linkers L B has the structure (LB1), (LB2) or (LB3): [ka]

[0098]

[0113] In the formula, n' is an integer ranging from 1 to 10, preferably from 1 to 4, and most preferably n'=2. * The wavy bond labeled Z 1 is connected to ** The ruffled junctions labeled as are linked to the BM.

[0099]

[0114] LC is a chain of at least 2, preferably 5 to 100, atoms selected from C, N, O, S, and P, which may typically be present in the DAR1 conjugate when y=1; L C The branch part is L M or (Z) z Connect to L C is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ and NR 21 wherein y' is 0, 1 or 2, preferably y'=2, and R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups.

[0100]

[0115] In a preferred embodiment, the antibody-conjugate is a DAR1 conjugate according to structure (2), where BM=N and the conjugate has the structure AB-[LD], i.e., x=y=1, and L has a structure selected from (L5)-(L7): [ka]

[0101]

[0116] In the formula, L 6 Both occurrences of are concatenated into a single AB, and L M is concatenated to the payload D.

[0102] [Linker L M ]

[0117] Linker L M is the structure -(L 1 ) n -(L 2 ) o -(L 3 ) p -(L 4 ) q -has L 1 , L 2 , L 3 and L 4 Z, L C or a linker connecting BM to D. Where L 1 , L 2 , L 3 and L 4 is a linker or linking unit, wherein each of n, o, p, and q is independently 0 or 1, and n+o+p+q is at least 1; p+q is 1 or 0, and o=p. In a preferred embodiment, the linker L 1 , L 2 and L 3 exists, and L 4 are absent (i.e., n=1; o=1; p=1; q=0). In another preferred embodiment, the linker L 1 and L 4 is present (i.e., n=1; o=0; p=0; q=1). In another preferred embodiment, the linker L 1only exists (i.e., n=1; o=0; p=0; q=0).

[0103] [Linker L 1 ]

[0118] Linker L 1 is either absent (n=0) or present (n=1). Preferably, the linker L 1 where n=1. L 1 is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 The alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may optionally be substituted, and the groups may optionally be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ and NR 21 wherein y' is 0, 1 or 2, preferably y'=2; 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups.

[0104]

[0119] In a preferred embodiment, the linker L 1contains a polar group. Such polar groups include (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,z'-diaminoalkanes (where z' is the number of carbon atoms in the alkane, preferably z'=1 to 10), -(O) a -C(O)-NH-S(O)2-NR 13 - (see structure (L1a) as further defined below), -C(S(O)3 (-) )-, -C(C(O)2 (-) )-, -S(O)2-, -P(O)2 (-) -, -O(CH2CH2O) t -, -NR 30 (CH2CH2NR 30 ) t -, and may be selected from the following two structures: [ka]

[0105]

[0120] With respect to the polar groups defined herein above, which terminus is Z 1 Which end is linked to (L 2 ) is irrelevant.

[0106]

[0121] The polar group may also contain an amino acid, preferably selected from Arg, Glu, Asp, Ser and Thr. 13 is further defined below for structure (L1a). t is an integer ranging from 0 to 15, preferably from 1 to 10, more preferably from 2 to 5, and most preferably t=2 or 4. Each R 30 are individually H, C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkal or C 1~12 Aralkyl. Linker L 1may contain two or more such polar groups, such as at least two polar groups. A polar group may also be a linker L branching from a branching moiety as defined elsewhere. 1 Preferably, nitrogen or carbon atoms are used as branching moieties. In the branching, -O(CH2CH2O) t The presence of polar groups is particularly preferred.

[0107]

[0122] In a preferred embodiment, the linker L 1 is or contains a sulfamide group, preferably a sulfamide group according to structure (L1a): [ka]

[0108]

[0123] The wavy lines represent the linkages, typically Q and L, to the remainder of the compound. 2 , L 3 or D, preferably Q and L 2 Preferably, ((O) a The C(O) moiety is linked to Q and NR 13 Part L 2 , L 3 or D, preferably L 2 is linked to.

[0109]

[0124] In structure (L1a), a=0 or 1, preferably a=1, and R 13 is hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24(Hetero)arylalkyl groups include O, S and NR 14 and optionally substituted and optionally interrupted by one or more heteroatoms selected from, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; 13 is preferably Sp as defined below, optionally via a spacer moiety; 2 and in one embodiment, D is linked to N via -(B) e -(A) f -(B) g is linked to N via —C(O)—; or R 13 is linked to another position in the linker, optionally via a spacer moiety, to form a cyclic structure. For example, R 13 may be linked to a linker via a CH2CH2 spacer moiety to form a piperazinyl ring, and the linkage to D is via the second nitrogen of the piperazinyl ring.

[0110]

[0125] In a preferred embodiment, R 13 is hydrogen, C1 to C 20 Alkyl groups, preferably C1-C 16 Alkyl groups, more preferably C1-C 10 is an alkyl group, or is optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker. As used herein, alkyl groups are defined as O, S and NR 14 , optionally substituted and optionally interrupted by one or more heteroatoms selected from, preferably O, and R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 13 is C1~C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 is an alkyl group, optionally interrupted by one or more O atoms, and optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment, R 13It is further preferred that R is a (poly)ethylene glycol chain containing a terminal -OH group. 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, even more preferably selected from the group consisting of hydrogen, methyl and ethyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker. Even more preferably, R 13 is hydrogen or is optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, most preferably R 13 is hydrogen.

[0111]

[0126] In a preferred embodiment, L 1 follows the structure (L1b): [ka]

[0112]

[0127] In the formula, a and R 13 is as defined above, and Sp 1 and Sp 2 is independently a spacer moiety, and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, more preferably b=0 and c=1. In one embodiment, the spacer Sp 1 and Sp 2 are independently straight-chain or branched C1 to C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group and C9-C 200 and arylalkynylene groups, wherein the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group are selected from the group consisting of O, S, and NR 16 and optionally substituted and optionally interrupted by one or more heteroatoms selected from the group 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. The alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, and preferably, the groups are interrupted by one or more O atoms and / or one or more S-S groups.

[0113]

[0128] More preferably, the spacer moiety Sp 1 and Sp 2 is, if present, independently a straight or branched chain C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylene group, C3-C 100 Cycloalkylene group, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkylarylene group, C7-C 100Aryl alkylene group, C8-C 100 Arylalkenylene group and C9-C 100 and arylalkynylene groups, wherein the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group are selected from the group consisting of O, S, and NR 16 and optionally substituted and optionally interrupted by one or more heteroatoms selected from the group 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0114]

[0129] Even more preferably, the spacer moiety Sp 1 and Sp 2 is, if present, independently a straight or branched chain C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylene group, C3-C 50 Cycloalkylene group, C5-C 50 Cycloalkenylene group, C8-C 50 Cycloalkynylene group, C7-C 50 Alkylarylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene group and C9-C 50 and arylalkynylene groups, wherein the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group are selected from the group consisting of O, S, and NR 16 and optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of:16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0115]

[0130] Even more preferably, the spacer moiety Sp 1 and Sp 2 is, if present, independently a straight or branched chain C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylene group, C3-C 20 Cycloalkylene group, C5-C 20 Cycloalkenylene group, C8-C 20 Cycloalkynylene group, C7-C 20 Alkylarylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene group and C9-C 20 and arylalkynylene groups, wherein the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group are selected from the group consisting of O, S, and NR 16 and optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of: 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0116]

[0131] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and are substituted with O, S, and NR 16 , optionally interrupted by one or more heteroatoms selected from the group of O, 16 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0117]

[0132] Most preferably, the spacer moiety Sp 1 and Sp 2 is, if present, independently a straight or branched chain C1-C 20 alkylene groups, wherein the alkylene groups are selected from the group consisting of O, S, and NR 16 and R 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is unsubstituted and is selected from the group consisting of O, S, and NR 16 , optionally interrupted by one or more heteroatoms, preferably selected from the group O and / or SS, and R 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0118]

[0133] Therefore, the preferred spacer moiety Sp 1 and Sp 2 As -(CH2) r -, -(CH2CH2) r -, -(CH2CH2O) r -, -(OCH2CH2) r-, -(CH2CH2O) r CH2CH2-, -CH2CH2(OCH2CH2) r -, -(CH2CH2CH2O) r -, -(OCH2CH2CH2) r -, -(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and 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.

[0119]

[0134] In a particularly preferred embodiment, L 1 is a linker for the DAR1 and DAR2 conjugate, x in structure (1) is 1, and -(W) k -(A) d -(B) e -(A) f -(B) g -(W) g -, wherein - d=0 or 1, preferably d=1; - e=an integer in the range of 0 to 10, preferably e=0, 1, 2, 3, 4, 5 or 6, preferably an integer in the range of 1 to 10, most preferably e=1, 2, 3 or 4; - f=0 or 1, preferably f=0; wherein d+e+f is at least 1, preferably in the range of 1 to 5; preferably d+f is at least 1, preferably d+f=1. - g=0 or 1, preferably g=1; - k=0 or 1, preferably k=1; A is a sulfamide group according to structure (L1a); - B represents a spacer, such as a PEG spacer. B represents L 2 , -(CH2) x -O- or -O-(CH2) x - individually selected from the parts; or (B) e is -((CH2) x -O) e1 -(CH2) x - moiety, where e1 is an integer ranging from 1 to 10, and each x is individually an integer ranging from 1 to 10. - W is -C(O)-, -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 Preferably, W is independently selected from -OC(O)NH-, -C(O)(CH2) m C(O)NH— or —C(O)NH—, where m is an integer ranging from 0 to 10, preferably m=0, 1, 2, 3, 4, 5 or 6, and most preferably m=2 or 3. In one embodiment, W is not C(O); - Preferably L 1 (W) k Through Q and L 2 , L 3 or linked to D, preferably (W) g More preferably, via C(O), and more preferably via C(O) 2 is linked to.

[0120]

[0135] L 1 In the context of this preferred embodiment, this is -(W) k -(A) d -(B) e -(A) f -(B) g -(C(O)) g -, where d, e, f, g, k, A, B and W are as defined above.

[0121]

[0136] In the context of this embodiment, the wavy line in structure (L1a) represents (W) k , (B) e and (C(O)) g A represents a linkage to an adjacent group such as structure (L1a) where a=1 and R 13 =H or C1~C 20 is an alkyl group, and more preferably R 13 =H or methyl, most preferably R 13 It is preferable to follow the formula (wherein =H).

[0122]

[0137] Preferred linkers L 1 is the structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein (a) k=0; d=1; g=1; f=0; B=—CH2—CH2—O—; e=1, 2, 3 or 4, preferably e=2. (b) k = 1; W = -C(O)(CH2) m C(O)NH-; m=2; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; f=0; g=1; e1=1, 2, 3 or 4, preferably e=1. (c) k=1; W=-OC(O)NH-; d=0; B=-CH2-CH2-O-; g=1; f=0; e=1, 2, 3 or 4, preferably e=2. (d) k = 1; W = -C(O)(CH2) m C(O)NH-; m=2; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; f=0; g=1; e1=1, 2, 3 or 4, preferably e1=4. (e)k=1;W=-OC(O)NH-;d=0;(B) e =-(CH2-CH2-O) e1 -CH2-CH2-; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4. (f)k=1;W=-(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, m=3; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4. (g) k=0; d=0; g=1; f=0; B=-CH2-CH2-O-; e=1, 2, 3 or 4, preferably e=2. (h) k=1; W=-C(O)NH-; d=0; g=1; f=0; B=-CH2-CH2-O-; e=1, 2, 3 or 4, preferably e=2.

[0123]

[0138] In this specification, when d and / or f=1, a=1 and R 13 Preferably, =H. Most preferably, the linker is structure (A).

[0124]

[0139] Alternatively, for DAR4 conjugates where x in structure (1) is 2, L 1 is particularly preferably represented as follows: -(W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A) d’ -(B) e’ -(A) f -(W) g’ -]2 wherein A, B, BM, W, d, e, f, g, and k are as defined above and are independently selected for each occurrence. As used herein, d', e', and g' are as defined for d, e, and g, respectively. 1 In the context of this preferred embodiment, this is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A) d’ -(B) e’ -(A)f -(C(O)) g’ -]2, where d, d', e, e', f, g, g', k, BM, A, B and W are as defined above.

[0125]

[0140] Preferred linkers L containing branched moieties 1 is the structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, wherein - k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A follows the structure ((L1a)) with a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2. k=d=g=e'=g'=1; f=d'=0; W=-C(O)-; B=B'=-CH2-CH2-O-; A is according to structure (L1a), a=0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0126] [Linker L 2 ]

[0141] Linker L 2 is a peptide spacer. Linker L 2 can be absent (o=0) or present (o=1). 2 may also be present in the capping group of a masked PBD dimer payload. 2 and a cleavable linker L 3 The combination of is well known in the art. The peptide spacer is (N(H)-HCR 17 -CO) n where R 17represents an amino acid side chain. As used herein, an amino acid may be a natural or synthetic amino acid. When the amino acid is proline, R 17 is linked to the nitrogen and (H) is absent. Preferably, the linker L 2 The L serves as a recognition and cleavage site for the cleavage enzyme, and more preferably, the peptide is recognized by a specific cleavage enzyme. This allows for cleavage in specific environments where these cleavage enzymes are expressed, such as in certain cancers. Since different peptide sequences are cleaved by different enzymes, the L 2 The groups also allow the ADC to be customized for a particular therapy. The peptide sequence can be cleaved by intracellular and / or extracellular enzymes.

[0127]

[0142] Preferably, all amino acids are in the L configuration. n is an integer ranging from 1 to 5, preferably from 2 to 5. Thus, the peptide spacer contains 1 to 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. Although any peptide spacer can be used, preferably the peptide spacer is Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly, Leu-Gly, Tyr-Gly,

[0049] The amino acid sequence is selected from 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-Gly-Gly-Phe, Phe-Phe-Gly-Gly, Gly-Gly-Phe-Phe, Gly-Gly-Gly-Phe-Gly and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn, and most preferably Val-Cit or Val-Ala. As used herein, AcLys is N-acetyl lysine and iGlu is isoglutamate. 2 In another embodiment, L 2 In another embodiment, L 2 In another embodiment, L 2 =Glu-Gly-Cit.

[0128]

[0143] R 17 represents an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, and more preferably those derived from the side chains of Val, Cit, Ala, Glu, and Lys. In other words, R 17 is preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred embodiments of R are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu) and CH(CH3)2(Val). Most preferably, R 17 is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0129]

[0144] In particularly preferred embodiments, the peptide spacer is represented by the general structure (L2): [ka]

[0130]

[0145] In the formula, R 17 is as defined above, preferably R 17 is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates (L 1 ) n and (L 3 ) p and preferably L according to structure (L3): 2 is via NH (L 1 ) n and (L 3 ) p is linked to.

[0131]

[0146] L 2 may contain specific peptide sequences that are cleavable by specific enzymes, which, if expressed or overexpressed only within the tumor microenvironment or within the endosomal / lysosomal compartments of tumor cells, increase the likelihood of targeted release of PBD dimers within tumors and reduced release in healthy tissues.

[0132] [Linker L 3 ]

[0147] Linker L 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is either absent (p=0) or present. 3 may also be present in the capping group of a masked PBD. 3 is present and p=1. 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L3): [ka]

[0133]

[0148] In the formula, the dashed line represents L 2 and D, and the PABC derivative is linked to L via NH. 2 is linked to.

[0134]

[0149] Ring A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. Ring A may be optionally substituted. In one embodiment, Ring A is selected from the group consisting of halogen, hydrophilic moieties HM, N(R 4 )2, C 1~4 and HM is as defined below, including preferred embodiments thereof. In preferred embodiments, the optional substituents are F, Cl, Br, OH, OR, 4 , SH, NH2, Et, Me and NO2. In particularly preferred embodiments, Ring A contains 0 to 2 substituents, more preferably 0 or 1 substituent, and most preferably Ring A is unsubstituted and contains NH and CR 21 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. In an alternative preferred embodiment, A is CH2-NMe-(Sar) n It is 1,3,4-phenyl having an -Ac group, where Sar refers to the repeating unit of polysarcosine, i.e., -C(O)-CH-N(CH)-; n is an integer ranging from 0 to 100, preferably from 1 to 50, more preferably from 5 to 20, and most preferably n=10; and Ac is acetyl.

[0135]

[0150] R 21 H, R 26 , C(O)OH and C(O)R 26 where R 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10(Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are O, S and NR 28 and optionally substituted and optionally interrupted by one or more heteroatoms selected from, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3 or 4. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0136]

[0151] L 2 The cleavage of linker L 3 This results in the 1,6-β-elimination of L, resulting in decarboxylation and release of the payload, D. 2 This advantageously increases the likelihood of payload release in regions where the enzyme is overexpressed. Furthermore, payload release may, in some cases, induce bystander killing, which is advantageous for tumors where not all cancer cells overexpress the target receptor.

[0137] [Linker L 4 ]

[0152] Linker L 4is either absent (p=0) or present (p=1). 4 may be present in the capping group. 3 and linker L 4 is typically a linker L M (p+q=1 or 0). 4 is a self-cleavable spacer, also called a self-immolative spacer. 4 is a para-glucuronide-meta-amido-benzyloxycarbonyl derivative, more preferably a derivative according to structure (L4): [ka]

[0138]

[0153] In the formula, the wavy line represents Q 1 or Z 1 or L 1 and to D. Typically, the moieties Q, Z are linked via NH. 1 or L 1 is linked to, preferably L 1 is linked to.

[0139]

[0154] A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Ring A is further defined above. Suitable 5-membered rings are oxazole, thiazole, and furan. Suitable 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,3,4-phenyl, 2,4,5-pyridyl, or 2,5,6-pyridyl. Most preferably, A is 1,3,4-phenyl.

[0140]

[0155] R 21 H, R 26 , C(O)OH and C(O)R 26 where R 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are O, S and NR 28 and optionally substituted and optionally interrupted by one or more heteroatoms selected from, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3 or 4. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0141]

[0156] Linker L 4 L 3 The linker L can be cleaved by β-glucuronidase similar to the mechanism of 4 ADCs containing L are particularly useful for the treatment of cancers with overexpression of β-glucuronidase. It has been reported that the concentration of β-glucuronidase in many solid tumors, including lung, breast, and gastrointestinal cancers, and in the tumor microenvironment is higher than in normal tissues, and this enzyme is not found in the systemic circulation. Therefore, L 4 It is preferred to use a conjugate according to the invention comprising:

[0142] [payload]

[0157] The payload (D) of the present invention is a masked PBD dimer that requires removal of the "mask" (capping group or cap) to release the active payload. Preferably, D conforms to any one of structures (D1) to (D4): [ka]

[0143]

[0158] where the wavy line represents the connection to the linker L; [ka] is in the pyrrolidine ring or between the pyrrolidine ring and D 2 represents a single or double bond between; dx is an integer ranging from 1 to 10, preferably dx is an integer ranging from 1 to 5, more preferably dx is 1 or 2, and most preferably dx is 1; D 1 and D 1 ' are each selected from H, OH, or SO2X; X is a halogen selected from fluorine, chlorine, bromine, or iodine; D 2 and D 2 ' are each selected from -H, -OH, =CH2 and -CH3; D 3 and D 3 ' are each selected from H, OH, OMe, SMe, NMe2, NO2, F, Cl, Br or I, preferably D 3 and D 3 Both of the ' are selected from OH, OMe or NMe2, most preferably D 3 and D 3 G and G' each represent a single bond or CH; J represents a single bond, CH, NH, -C≡C-, -C≡C-CH-, or O; C 1 and C 2 is a capping group.

[0144]

[0159] Preferably, D conforms to structure (D2), and more preferably (D2) conforms to structure (D21): [ka]

[0145]

[0160] Even more preferably, D is according to structure (D21) where dx=1 or dx=2, most preferably dx=1.

[0146]

[0161] In another preferred embodiment, D conforms to structure (D22): [ka]

[0147]

[0162] Even more preferably, D is according to structure (D22) where dx=1 or dx=2, most preferably dx=2.

[0148] [Capping group]

[0163] The conjugates according to the present invention comprise at least one capping group. In structures (D1) to (D4), (D21) and (D22), C 1 and C 2 represents a capping group. The capping group comprises a self-immolative spacer. More preferably, C 1 and C 2 is the structure-L 4 -HM, -L 3 -L 2 -HM, (C1), (C2) and (C3) are independently selected from: [ka]

[0149]

[0164] wherein HM is a hydrophilic moiety. The hydrophilic moiety is a moiety that increases solubility in aqueous solution. Preferably, the hydrophilic moiety is selected from a sulfonated side chain, a carbamoylsulfamide side chain, a polysarcosine-containing side chain, or a polyethylene glycol-containing side chain; more preferably, the hydrophilic moiety is selected from a carbamoylsulfamide side chain or a polyethylene glycol side chain; most preferably, the hydrophilic moiety is selected from a polyethylene glycol side chain. Alternatively, the hydrophilic moiety is a chain of at least two atoms independently selected from C, O, S, and P; more preferably, the hydrophilic moiety is -(L 1 ) c’ -R 23 , -C(C 23 )2-(L 1 ) c’ -R 23 and most preferably the hydrophilic moiety is selected from the structures (HM1), (HM2), (HM3), (HM4) and HM5: [ka]

[0150]

[0165] In the formula, L 1 , C 23 and c' are as defined above, and each R 23 are independently hydrogen and C1-C6 alkyl, cx is an integer of 1 to 5, more preferably cx is 1 or 2, c' is 0 or 1, and preferably c'=1.

[0151]

[0166] It is particularly preferred that the HM at (C3) is linked to the disulfide via a carbon atom, said carbon atom being at least one C 23 More preferably, HM in structure (C3) is structure (HM4) or (HM5), most preferably (HM5).

[0152]

[0167] Q is selected from O, NH, NMe, or N-HM, preferably Q is O or NMe, most preferably Q is NMe.

[0153]

[0168] A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,4-phenyl, 2,5-pyridyl or 3,6-pyridyl. Most preferably, A is 1,4-phenyl, where C 22 is linked to the 2-position.

[0154]

[0169] C 21 is H, C 26 , C(O)OH and C(O)C 26 is selected from, where C 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are O, S and NC 28 and optionally substituted and optionally interrupted by one or more heteroatoms selected from C 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3 or 4. More preferably, C 21 is H or C(O)R 26 where C 26= 4-methyl-piperazine or morpholine. Most preferably, C 21 is H.

[0155]

[0170] C 22 is selected from HM, NO2 or H, more preferably C 22 is selected from NO2 or H.

[0156]

[0171] C 23 is H or a C1-C3 alkyl group, preferably C 23 is H or methyl, more preferably C 23 is H.

[0157] Preferred Embodiment

[0172] In one embodiment, the linker does not contain a self-immolative spacer. The advantage of using a conjugate without a self-immolative spacer is that when the conjugate is degraded in the lysosome, the payload remains linked to the charged moiety and therefore cannot escape from the cell. Unlike the linker, the cap is cleavable, so the payload has sufficient potency. This embodiment is particularly advantageous for the treatment of cancers where very low bystander killing is desired, because bystander killing also affects healthy cells. Therefore, conjugates containing non-self-immolative linkers are preferred for use in the treatment of cancers selected from liquid cancers or solid cancers that homogeneously express the target receptor, and more preferably, conjugates containing non-cleavable linkers are used for the treatment of liquid cancers.

[0158]

[0173] In a preferred embodiment, the conjugate according to the present invention comprises a linker comprising a self-immolative spacer. The advantage of using a cleavable linker is that bystander killing can be induced. The conjugate according to the present invention may have the same cleavage mechanism for the linker as for the cap, which advantageously allows for immediate release of the payload in its most potent form. Alternatively, the cleavage mechanisms for the cap and the linker are different. The advantage of having different cleavage mechanisms for the linker and the cap is that the cap is released at a different stage from the payload, which can increase the tolerability of the conjugate. In a preferred embodiment, the cap is cleaved under extracellular conditions, while the linker is cleaved under intracellular conditions.

[0159]

[0174] In a preferred embodiment, the conjugate comprises a payload according to structure (D1) or (D2), wherein the conjugate comprises a L M More preferably, L M The self-immolative linker of C is cleaved by a mechanism separate from the self-immolative spacer of the cap. 1 -L 4 -HM, (C2) or (C3), and L M includes (L6) and (L7), most preferably C 1 is (C3).

[0160]

[0175] In another preferred embodiment, L M is C 1 Same as, and if present, C 2 More preferably, L comprises a self-immolative linker that is cleaved by the same mechanism as M , C 1 , and if present, C 2 L 2 and L 3 and even more preferably, L M , C 1 , and if present, C 2 L 2 and L 3where L 2 is selected from Glu-Gly-Cit, Glu-Gly-Val, Ala-Asn, Asn-Ala, Pro-Leu-Gly, Asn-Asn, Glu-Val-Ala, (D-Leu)-Ala-Glu, and most preferably L 2 is Glu-Gly-Cit or Asn-Ala.

[0161]

[0176] In a preferred embodiment, the conjugate is a DAR1 or DAR2 conjugate, and L M -D has a structure selected from the group consisting of (LM1) to (LM4): [ka] In the formula, the wavy line represents Z 1 Indicates a connection to

[0162]

[0177] In another preferred embodiment, the antibody-conjugate is a DAR4 conjugate having a linker-payload moiety according to structure (L5) or (L6): [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , L 4 , o, q and D are as defined above.

[0163]

[0178] More preferably, the linker-payload moiety is according to (LM7) or (LM8): [ka]

[0164]

[0179] In a preferred embodiment, the conjugate according to the present invention is selected from structures (4) to (7): [ka] [ka]

[0165]

[0180] Preferably, L 2 The cap is Glu-Gly-Cit, Glu-Gly-Val, Val-Cit, Val-Ala, Val-Lys, Val-Arg, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-C it, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Asn-Ala, Phe-Phe, Gly-Gly, Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, Phe-Gly, Phe-G The amino acid sequence of the present invention is selected to include a peptide spacer selected from the group consisting of Ac-Lys, Ser-Gly, Gly-Phe-Gly, more preferably Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Asn-Asn, Asn-Ala, Glu-Val-Ala or Glu-Gly-Cit, more preferably Val-Cit, Val-Ala, Asn-Ala or Glu-Gly-Cit, most preferably Val-Cit or Val-Ala. As used herein, AcLys is eN-acetyl lysine and iGlu is isoglutamate. In one embodiment, L 2 In one embodiment, L 2 =Val-Ala.

[0166]

[0181] In a preferred embodiment, the conjugate conforms to structure (8): [ka]

[0167]

[0182] More preferably, the conjugate comprises a linker L in the cap according to structure (8): 2is selected from Glu-Gly-Cit, Glu-Gly-Val, Ala-Asn, Asn-Ala, Pro-Leu-Gly, Glu-Val-Ala or Asn-Asn. Most preferably, the linker L in the cap 2 is selected from Glu-Gly-Cit, Glu-Gly-Val, Ala-Asn, Asn-Ala, Pro-Leu-Gly, or Asn-Asn.

[0168]

[0183] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (30): [ka]

[0169]

[0184] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (31): [ka]

[0170]

[0185] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (32): [ka]

[0171]

[0186] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (33): [ka]

[0172]

[0187] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (34): [ka]

[0173]

[0188] In another preferred embodiment, the conjugate can be obtained by reacting an azide or tetrazine modified antibody with a linker drug construct according to structure (35): [ka]

[0174]

[0189] Preferably, the conjugate according to the present invention can be obtained by reacting a linker-drug construct according to any one of structures (30) to (35) with a modified antibody.

[0175] [medical care]

[0190] In a second aspect, the present invention relates to a conjugate according to the invention for use as a medicament. More preferably, the conjugate is used for the treatment of cancer.

[0176]

[0191] The present invention further relates to a pharmaceutical composition comprising an antibody-payload conjugate according to the invention and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is used in medical treatment, more preferably, the pharmaceutical composition is used in the treatment of cancer.

[0177]

[0192] The inventors have surprisingly found that the antibody-drug conjugates according to the present invention are superior to conventional masked PBD conjugates in terms of safety and / or efficacy, such that the therapeutic index of the antibody-drug conjugates according to the present invention is increased compared to conventional masked PBD antibody-conjugates. Preferably, increasing the therapeutic index of the antibody-conjugate is selected from the following: a) increasing the therapeutic effect of the antibody-conjugate; and / or b) Increasing the tolerability of the antibody-conjugate.

[0178]

[0193] The increased therapeutic efficacy of an antibody-conjugate according to the invention may take the form of a reduction in tumor size and / or prolonged regression when compared to conventional masked PBD ADCs. The increased tolerability of an antibody-conjugate according to the invention may take the form of a reduction in signs of toxicity when compared to administration of a masked PBD ADC made by conventional techniques. Reduced signs may also be referred to as a reduction in symptoms or side effects of cancer treatment and may involve one or more clinical signs, such as reduced weight, reduced mobility, reduced food intake, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology.

[0179] [Process for the synthesis of antibody-drug conjugates]

[0194] In a third aspect, the present invention relates to a process for preparing an antibody-drug conjugate according to the present invention. The process according to the present invention comprises the steps of: 6 -F 1 ) x The method includes reacting the modified antibody of formula (I) with a linker-drug construct. The reaction is a conjugation reaction, which forms a covalent bond between the masked PBD payload and the antibody. The reaction is a metal-free click reaction, which forms an antibody-drug conjugate, where the drug is linked to a linker-drug construct. 1 and F 1 Linking group Z formed by metal-free click reaction between 1 Preferably, the metal-free click reaction is a 1,3-dipolar cycloaddition.

[0180]

[0195] In the process according to the invention, the structure AB-(L 6 -F 1 ) x a modified antibody of the formula: 1 is the linker, and F 1 is the Q in the metal-free click reaction 1where x is an integer ranging from 1 to 8. 1 Q of the linker-drug construct according to structure (9) or (10) 1 By reacting with 1 In one embodiment, the modified antibody is AB-(F 1 ) x 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 documents, conjugation reactions between modified antibodies and linker-drug constructs containing cytotoxins are known to those skilled in the art.

[0181]

[0196] The process according to the present invention comprises the following steps: i) The antibody containing y core N-acetylglucosamine (GlcNAc) moieties is prepared by the method of formula S(F 1 ) x -P(wherein, S(F 1 ) x is x reactive groups F that can react with reactive group Q 1 and P is a nucleoside mono- or diphosphate) and a catalyst (the catalyst is S(F 1 ) x and contacting the antibody according to formula (26) with a nucleotide sequence (which can transfer a nucleotide to the core-GlcNAc moiety) to obtain an antibody according to formula (26): AB-[GlcNAc(Fuc) w -SCIENCE FICTION 1} x ] y (26) (In the ceremony Fuc is fucose; w is 0 or 1; x is 1 or 2, preferably x is 1; y is 1 or 2, preferably y is 2; ii) optionally a compound of the formula AB-[GlcNac(Fuc)wS{F 1}] y wherein y=2, and reacting the modified antibody with a compound of formula (27): [ka] Obtaining a modified antibody according to formula (28); [ka] iii) reacting the modified antibody according to formula (26) or (28) with a compound selected from structures (9), (10) and (11) to obtain an antibody-conjugate according to structure (1). [ka] wherein if optional step ii) is performed, the compound used in step iii) is represented by structure (11); and if optional step ii) is not performed, the compound used in step iii) is represented by structure (9) or (10).

[0182] [Metal-free click reaction]

[0197] Metal-free click reactions are well known in the art (see, e.g., WO 2014 / 065661 and Nguyen and Prescher, Nature rev. 2020, doi:10.1038 / s41570-020-0205-0, both of which are incorporated by reference) and can typically take the form of a 1,3-dipolar cycloaddition or a (4 + 2) cycloaddition. The alkyne-azide cycloaddition can be strain-promoted (e.g., strain-promoted alkyne-azide cycloaddition, SPAAC). In preferred embodiments, the bioconjugation reaction is a metal-free strain-promoted cycloaddition, most preferably a metal-free strain-promoted alkyne-azide cycloaddition. In preferred embodiments, the conjugation is achieved via a cycloaddition, such as a (4 + 2) cycloaddition or a 1,3-dipolar cycloaddition, preferably a 1,3-dipolar cycloaddition.

[0183]

[0198] A typical (4+2) cycloaddition is the Diels-Alder reaction, where Q 1 is a diene or dienophile. As one skilled in the art will appreciate, the term "diene" in the context of the Diels-Alder reaction refers to 1,3-(hetero)dienes, including (R2C=CR-CR=CR2), imines (e.g., R2C=CR-N=CR2 or R2C=CR-CR=NR, R2C=NN=CR2), and carbonyls (e.g., R2C=CR-CR=O or O=CR-CR=O). Hetero-Diels-Alder reactions with N- and O-containing dienes are known in the art. Any diene known in the art suitable for a (4+2) cycloaddition can be converted to a reactive group Q. 1 Preferred dienes include tetrazines, 1,2-quinones, and triazines. Any dienophile known in the art suitable for (4+2) cycloaddition can be used in combination with the reactive group Q 1 Although Q can be used as a dienophile, the dienophile is preferably an alkene or alkyne group as described above, most preferably an alkyne group. For conjugation via (4+2) cycloaddition, Q 1 is a dienophile (and F 1 is preferably a diene), and more preferably, Q 1 is or contains an alkynyl group.

[0184]

[0199] In the case of 1,3-dipolar cycloaddition, Q 1 is a 1,3-dipole or dipolarophile. Any 1,3-dipole known in the art suitable for 1,3-dipolar cycloaddition can be substituted with the reactive group Q. 1 Preferred 1,3-dipoles include azide groups, nitrone groups, nitrile oxide groups, nitrile imine groups, and diazo groups. Any dipolarophile known in the art suitable for 1,3-dipolar cycloaddition can be used as a reactive group Q. 1Although the dipolarophile can be used as a dipolarophile, preferably the dipolarophile is an alkene or alkyne group, most preferably an alkyne group. For conjugation via 1,3-dipolar cycloaddition, Q 1 is the dipolarophile (and F 1 is preferably a 1,3-dipole), and more preferably, Q 1 is or contains an alkynyl group.

[0185]

[0200] Therefore, in a preferred embodiment, Q 1 is selected from dipolarophiles and dienophiles.

[0186] [Click Probe Q 1 ]

[0201] Click Probe Q 1 The linker-drug construct is represented by the structure AB-(F 1 ) x It is used in conjugation reactions to link to antibodies of Q 1 is the metal-free click reaction of click probe F 1 Such click probes are known in the art and include cyclic alkenes, cyclic alkynes, azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and sydnones. Preferably, Q 1 is a cyclic alkene or cyclic alkyne moiety, most preferably Q 1 is a cyclic alkyne moiety.

[0187]

[0202] Therefore, in a particularly preferred embodiment, Q 1contains a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, wherein the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group, or a (hetero)cyclodecynyl group. In the present specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, wherein the (hetero)cyclooctynyl group is optionally substituted.

[0188]

[0203] In a particularly preferred embodiment, Q 1 contains a (hetero)cycloalkynyl group or a (hetero)cycloalkenyl group, and has the structure (Q 1 ) according to: [ka]

[0189]

[0204] During the ceremony: - Bonds shown as [ka] is a double or triple bond; R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24and (hetero)arylalkyl groups, wherein the alkyl group, the (hetero)aryl group and the (hetero)arylalkyl group are optionally substituted and include two substituents R 15 are joined together to form an optionally substituted cyclized cycloalkyl or an optionally substituted cyclized (hetero)arene substituent, and R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked 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 is an integer in the range of 0 to 16.

[0190]

[0205] Typically, v = (u + u') × 2 (the connection to L indicated by the wavy bond is Y 2 (when the connection to L represented by the wavy bond is via one of the carbon atoms) or [(u+u')×2]-1 (when the connection to L represented by the wavy bond is via one of the carbon atoms).

[0191]

[0206] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and conforms to structure (Q1a): [ka] (In the formula, R 15 and Y 2 is as defined above; u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4 or 5, where u+u'=4, 5, 6, 7 or 8; v = an integer in the range 8 to 16). In a preferred embodiment, u+u'=4, 5 or 6, more preferably u+u'=5. In a preferred embodiment, v=8, 9 or 10, more preferably v=9 or 10, and most preferably v=10.

[0192]

[0207] In a preferred embodiment, Q 1 is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) to (Q20) and (Q38a) shown herein below. [ka]

[0193]

[0208] where the connection to L is represented by a wavy bond, Q 1 The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) may have a linkage to L, or may contain a hydrogen atom, or may be optionally functionalized. (-) is preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably, B (-) teeth, (-) OTf. In the conjugation reaction, B (-) will be exchanged with anions present in the reaction mixture anyway, so B (-) is not required to be a pharmaceutically acceptable anion. 1When used in, the negatively charged counterion is preferably pharmaceutically acceptable upon isolation of the conjugate according to the invention so that the conjugate can be readily used as a drug.

[0194]

[0209] In a further preferred embodiment, Q 1 is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38a) shown herein below. [ka]

[0195]

[0210] In structure (Q38), B (-) is preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably, B (-) teeth, (-) It is OTf.

[0196]

[0211] In a preferred embodiment, Q 1 preferably comprises a (hetero)cyclooctyne or (hetero)cycloheptyne moiety according to the structures (Q8), (Q26), (Q27), (Q28), or (Q37), which are optionally substituted. Each of these preferred options for Q is further defined herein below.

[0197]

[0212] Therefore, in a preferred embodiment, Q 1 comprises a heterocycloheptyne moiety according to structure (Q37), also referred to as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is unsubstituted.

[0198] In an alternative preferred embodiment, Q 1comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q 1 is preferably a (hetero)cyclooctyne moiety according to the structure (Q39) shown below, where V is (CH) l where l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably, l is 0, 1 or 2, and most preferably, l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q 1 conforms to structure (Q42), further defined below.

[0199]

[0213] In an alternative preferred embodiment, Q 1 comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also referred to as a DIBO, DIBAC, DBCO or ADIBO group, which is optionally substituted. In the context of this embodiment, Q 1 is preferably a (hetero)cyclooctyne moiety according to the structure (Q40) or (Q41) shown below, wherein Y 1 is O or NR 11 and R 11 are independently hydrogen, straight or branched chain C1-C 12 Alkyl group or C4-C 12 (Hetero)aryl groups. The aromatic ring of (Q40) is optionally O-sulfonylated at one or more positions, while the ring of (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q 1 conforms to structure (Q43), further defined below.

[0200]

[0214] In an alternative preferred embodiment, Q 1 contains a heterocycloheptynyl group and conforms to structure (Q37). [ka]

[0201]

[0215] In a particularly preferred embodiment, Q 1 contains a (hetero)cyclooctynyl group and conforms to structure (Q43): [ka]

[0202]

[0216] In a particularly preferred embodiment, Q 1 contains a cyclooctynyl group and conforms to structure (Q42): [ka] (In the formula, R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups; R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is a second occurrence of Q or D linked via a spacer moiety; where l is an integer ranging from 0 to 10.

[0203]

[0217] In a preferred embodiment of the reactive group according to structure (Q42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 15 is hydrogen or C1-C6 alkyl, and more preferably, R 16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 19is H. In preferred embodiments of the reactive group according to structure (Q42), I is 0 or 1, and more preferably, l is 1.

[0204]

[0218] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and conforms to structure (Q43): [ka]

[0205]

[0219] During the ceremony: R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 and (hetero)arylalkyl groups, wherein the alkyl group, the (hetero)aryl group and the (hetero)arylalkyl group are optionally substituted and include two substituents R 15 are joined together to form an optionally substituted cyclized cycloalkyl or an optionally substituted cyclized (hetero)arene substituent, and R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; Y is N or CR 15 is.

[0206]

[0220] In a preferred embodiment of the reactive group according to structure (Q43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16is hydrogen or C1-C6 alkyl, and more preferably, R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of the reactive group according to structure (Q43), Y is N or CH, more preferably Y=N.

[0207]

[0221] In an alternative preferred embodiment, Q 1 contains a cyclic alkene moiety. 1 may also be referred to as a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted. Particularly preferred are a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctenyl group, wherein the (hetero)cyclopropenyl group, the trans-(hetero)cycloheptenyl group, or the trans-(hetero)cyclooctenyl group is optionally substituted. Preferably, Q 1 includes a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). 3 is C(R 23 )2, NR 23 or O, and each R 23 are individually hydrogen, C1-C6 alkyl, or optionally bonded to L via a spacer and labeled [ka] is a single or double bond. In a further preferred embodiment, the cyclopropenyl group conforms to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group conforms to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group conforms to structure (Q52), (Q53), (Q54), (Q55), or (Q56). [ka]

[0208]

[0222] In the formulae, the R group on Si in (Q50) and (Q51) is typically alkyl or aryl, preferably C1 to C6 alkyl.

[0209]

[0223] Q 2 is F 2 reacts with a further linking group Z 2 Z is a group that forms 2 Preferred options for Q are defined above. In a particularly preferred embodiment, Q 2 Q 1 and Q is selected from the same preferred embodiments as 1 and Q 2 are selected individually. If the compound is, for example, (10) or (27), two groups Q 1 When it comprises both groups, it is preferred that both groups are the same.

[0210] [Reactive moiety F 1 ]

[0216] F 1 is the Q in the conjugation reaction 1 and the conjugation reaction is a cycloaddition. As will be appreciated by those skilled in the art, F 1 The answer is Q 1 The same as the choices in F 1 and Q 1 are reactive with each other. Therefore, F 1comprises a click probe. Click probes are reactive to cycloaddition (click reaction) and are preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, or sydnones, and most preferably an azide. In a preferred embodiment, F 1 is an azide.

[0211] Two or more reactive groups F 1 may be present in the antibody. 1 may be naturally occurring or may be placed in the antibody by specific techniques, such as (bio)chemical or recombinant genetic techniques. Reactive groups placed in the antibody are prepared by chemical synthesis, for example azides or terminal alkynes. 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 documents, the conjugation reaction between modified antibodies and linker-toxin-constructs is known to those skilled in the art.

[0212] Preferably, F 1 is a click probe reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, and is typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and sydnones. Preferred structures for the reactive group are structures (F1) to (F10) shown herein below. [ka]

[0213]

[0219] In the formula, the wavy bond represents a link to a payload. For (F3), (F4), (F8) and (F9), the payload can be linked to any one of the wavy bonds. Then, the other wavy bond can be linked to a hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 sulfonyl groups, each of which (excluding hydrogen) is O, S, and NR 32 and R 32 are independently selected from hydrogen and C1-C4 alkyl groups. Those skilled in the art will recognize which R groups are groups F 1 For example, the R group attached to the nitrogen atom of (F3) can be selected from alkyl and aryl, and the R group attached to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl. Preferably, the reactive group F 1 is selected from azide or tetrazine. Most preferably, the reactive group F 1 is an azide.

[0214]

[0220] F 2 Q 2 reacts with a further linking group Z 2 Z is a group that forms 2 Preferred options for are defined above. In a particularly preferred embodiment, F 2 is F 1 and selected from the same preferred embodiments as 1 and F 2 are selected individually.

[0215] [Obtaining modified antibodies] The modified antibody can be prepared by combining an antibody containing y core N-acetylglucosamine (GlcNAc) moieties with a formula S(F) x -P(wherein, S(F1 ) is a group consisting of x reactive groups F capable of reacting with reactive group Q. 1 and P is a nucleoside mono- or diphosphate) and a catalyst (the catalyst is S(F 1 ) moiety to the core-GlcNAc moiety) to obtain an antibody according to formula (26): AB-[(L 6 )-(F 1 ) x ] y (26) (In the ceremony AB is a cell binding agent; b is 0 or 1; L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, where G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is —N(H)C(O)CH—, —N(H)C(O)CF—, or —CH—; F 1 is a reactive moiety; x is 1 or 2; and y is 1 or 2.

[0216] In one embodiment, the modified antibody according to structure (26) is reacted with a compound according to structure (27): [ka] Obtaining the modified antibody according to structure (28): [ka]

[0217]

[0223] In a preferred embodiment, the modified antibody according to structure (26) is reacted with a compound according to structure (9) to give a DAR2 or DAR4 conjugate, or with structure (10) to give a DAR1 conjugate. [ka]

[0218]

[0224] In another preferred embodiment, a modified antibody according to structure (28) is reacted with a compound according to structure (11): F 2 -L M -(D) x (11)

[0219]

[0225] A fourth aspect of the present invention relates to precursors that can be used in the synthesis of antibody-drug conjugates, which precursors conform to structure (9), (10) or (11). [ka]

[0220]

[0226] In the formula, F 2 Q 2 is the moiety that can react with Q 1 , L B ,BM,(L C ) c’ , (Z) Z’ , L M and D are as defined above. D, L as described for the conjugate M , Z 1 and Z 2 A preferred embodiment of the present invention is the precursor D, L M and Q 1 , Q 2 , F 1 and F 2 The same is also preferred for the group.

[0221]

[0227] In preferred embodiments, precursors of the present invention conform to structure (9) or (10), more preferably precursors of the present invention conform to structure (9) where x=1.

[0222] [Application]

[0228] The conjugates according to the present invention are particularly suitable for the treatment of cancer. Compounds according to structure (1) are further suitable for killing cells. In this respect, the present invention also relates to the use of the conjugates according to the present invention for killing cells, as well as to a method for killing cells, comprising contacting a cell with a conjugate according to the present invention. The use and method are typically ex vivo or in vitro.

[0223]

[0229] The conjugates of the present invention are particularly suitable for the treatment of cancer. In this regard, the present invention further relates to a method for the treatment of cancer, comprising administering a conjugate according to the present invention to a subject in need thereof. The subject in need thereof 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 in this regard. The described method is typically suitable for the treatment of cancer. In the method according to this embodiment, the antibody-conjugate is typically administered in a therapeutically effective dose. This embodiment of the present invention can also be expressed as a conjugate according to the present invention for use in the treatment of cancer. In other words, this embodiment relates to the use of a conjugate according to the present invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of cancer. In this context, the treatment of cancer is assumed to include treating, imaging, diagnosing, preventing tumor growth, inhibiting tumors, and reducing tumors.

[0224]

[0230] This aspect of the present invention can also be described as a method for targeting tumor cells expressing a specific extracellular receptor, comprising contacting a conjugate according to the present invention with cells that may possibly express the extracellular receptor, and wherein the antibody specifically targets the extracellular receptor. Thus, the method according to this aspect is suitable for determining whether a cell expresses a desired extracellular receptor. These tumor cells may be present in a subject, in which case the method comprises administering a conjugate according to the present invention to a subject in need thereof. Alternatively, the method is performed ex vivo or in vitro. In a preferred embodiment, the cells that may possibly express the extracellular receptor are cells that express the extracellular receptor. Targeting tumor cells preferably includes one or more of treating, imaging, diagnosing, preventing growth, inhibiting, and reducing tumor cells.

[0225]

[0231] In the context of diagnosis, it is generally unclear whether the contacted cells actually express the specific extracellular receptor that is being investigated.For example, in the diagnosis of HER2-positive breast cancer, the conjugate that contains the antibody that targets HER2, such as trastuzumab, can be contacted with cells.If tumor cells actually express HER2, the conjugate will target cells, but if tumor cells do not express HER2, the conjugate will not target cells.Similarly, in the treatment of cancer cells that specifically express extracellular receptor, those skilled in the art will understand that a cell binding agent such as an antibody that targets this specific extracellular receptor should be used.

[0226]

[0232] In the methods of the present invention, the extracellular receptor may be 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, Brevican, c-kit, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD 45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, D PEP3, 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, guanylyl 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, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema Preferably, the extracellular receptor is selected from the group consisting of 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tenascin. Likewise, preferably, the tumor cells express an extracellular receptor selected from the same group.One skilled in the art can match a desired extracellular receptor with an appropriate cell binding agent that can target that extracellular receptor.

[0227]

[0233] In this respect, the present invention also relates to a pharmaceutical composition comprising a conjugate according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains a conjugate according to the present invention in a pharmaceutically effective dose. In a preferred embodiment, the conjugate according to the present invention is present in the pharmaceutical composition in a dose of 100 μg to 10 mg per kg of body weight of the subject to be treated.

[0228]

[0234] The present inventors have unexpectedly found that conjugates according to the present invention are superior to conventional conjugates containing PBD-derived toxins in terms of safety and / or efficacy, such that the therapeutic index of the antibody-conjugates according to the present invention is increased compared to conventional PBD-containing conjugates. Considering the reduced toxicity of compounds according to structure (1), the safety of the conjugates according to the present invention is particularly improved. Therefore, higher doses of the conjugates can be administered to subjects in need thereof, which in turn has further benefits in treatment. Conventional conjugates of PBDs and cell-binding agents, such as antibodies, must be administered at very low doses, and as a result, administration of excessively high doses is not uncommon. This can lead to specific cell death and, consequently, undesirable side effects of cancer treatment. Furthermore, administration of PBD-antibody conjugates at these conventional low doses adversely affects biodistribution, resulting in less efficient tumor targeting. Thus, the present inventors have found that masked PBD dimer conjugates according to the present invention have reduced toxicity, resulting in improved therapeutic index, particularly safety or tolerability. Improved therapeutic efficacy of the conjugates according to the invention may take the form of reduced tumor size and / or prolonged regression when compared to conventional conjugates. Increased tolerability of the conjugates according to the invention may take the form of reduced signs of toxicity when compared to administration of conventional conjugates. Reduced signs may also be referred to as reduced symptoms or side effects of cancer treatment and may involve one or more clinical signs, such as reduced weight, reduced mobility, reduced food intake, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology.

[0229] [Example]

[0235] The invention is illustrated by the following examples. [BCN-Linker Synthesis] [ka]

[0230] Example 1: Synthesis of Compound A

[0236] To a solution of BCN-OH (3.0 g, 20 mmol) in DCM (300 mL) was added chlorosulfonyl isocyanate (1.74 mL, 2.83 g, 20 mmol) under a N atmosphere. After stirring at ambient temperature for 15 min, EtN (5.58 mL, 4.0 g, 40 mmol) and 2-(2-aminoethoxy)ethanol (2.2 mL, 2.31 g, 22 mmol) were added. The mixture was stirred for 15 min and quenched by adding aqueous NH4Cl (sat., 300 mL). After separation, the aqueous layer was extracted with DCM (200 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated. The residue was purified by column chromatography on silica gel (0% to 10% MeOH in DCM) to give A as a yellowish oil (4.24 g, 11.7 mmol, 58%). 1 H NMR(400MHz,CDCl3)δ(ppm)6.0(bs,1H),4.30(d,J=8.2Hz,2H),3.79-3.74(m,2H),3.67-3.62(m,2H),3.61-3.57 (m,2H),3.35(t,J=4.9Hz,2H),2.37-2.15(m,6H),1.63-1.49(m,2H),1.40(quintet,J=8.7Hz,1H),1.05-0.93(m,2H).

[0231] [Example 2. Synthesis of Compound B]

[0237] To a solution of TA (708.4 g, 1.34 mmol, 1.0 equiv) in anhydrous DMF (1.0 mL) was added bis(pentafluorophenyl)carbonate (583.4 g, 1.48 mmol, 1.1 equiv) and DiPEA (0.7 mL, 519.0 mg, 4.01 mmol, 3.0 equiv). After stirring at ambient temperature for 3.5 h, the reaction mixture was diluted with DCM (1.5 mL) and purified by flash column chromatography on silica gel (0% to 10% acetone in DCM) to give B as a clear orange oil (459.7 mg, 0.82 mmol, 61%). 22 H 24 F5N2O8S + (M+H + LCMS (ESI+) calculated for 571.49, found 571.40. [PBD-linker drug] [ka]

[0232] [Example 3. Synthesis of Compound D]

[0238] To a solution of Alloc-PBD derivative C (10.9 mg, 7.46 μmol, 1 equiv., commercially available from MedChemExpress) in anhydrous DCM (500 μL, degassed with N2) was added pyrrolidine (1.5 mg, 1.8 μL, 22.0 μmol, 2.9 equiv.) and Pd(PPh3)4 (22.5 μL, 9.95 mM, 0.22 μmol, 0.03 equiv.). After 35 min at ambient temperature, the reaction mixture was diluted with DCM (2.0 mL) and washed with aqueous NH4Cl (sat., 1.5 mL). The two layers were separated, and the aqueous layer was extracted with DCM (2 × 2 mL). The combined organic layers were dried (Na2SO4) and directly purified by flash column chromatography on silica gel (0% → 20% MeOH in DCM) to give compound D as a yellow solid (8.0 mg, 6.0 μmol, 80%). C 69 H 89 N 10 O 20 + (M+H + LCMS (ESI+) calculated for 1378.50, found 1378.01.

[0233] Example 4: Synthesis of Compound E

[0239] To a solution of D (8.0 mg, 6.0 μmol, 1.0 equiv) in anhydrous DCM (300 μL) was added a stock of B in DCM (9.0 mg, 140 μL, 16.0 μmol, 116 mM, 2.7 equiv) and EtN (2.0 mg, 2.0 μL, 20 μmol, 3.0 equiv). After stirring at ambient temperature for 6 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% to 10% MeOH in DCM) to give E as an off-white solid (5.6 mg, 3.17 μmol, 54%). 58 H 110 N 12 O 27 S + (M+H+ LCMS (ESI+) calculated for 1764.92, found 1764.44.

[0234] Example 5: Synthesis of Compound H [ka]

[0240] To a solution of Alloc-PBD derivative F (20.0 mg, 14.0 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (600 μL, degassed with N) was added pyrrolidine (2.9 mg, 3.4 μL, 41.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (31.0 μL, 17.4 mM, 1.23 μmol, 0.09 equiv.). After 60 min at ambient temperature, the reaction mixture was diluted with DCM (4.0 mL) and washed with aqueous NH4Cl (sat., 2.5 mL). The two layers were separated, and the aqueous layer was extracted with DCM (2 × 2 mL). The combined organic layers were dried (Na2SO4), filtered, and directly purified by flash column chromatography on silica gel (0% → 35% MeOH in DCM) to give G as an off-white solid (8.9 mg, 6.4 μmol, 47%). C 68 H 86 N 11 O 21 + (M+H + LCMS (ESI+) calculated for 1393.47, found 1393.41.

[0235]

[0241] To a solution of G (8.9 mg, 6.4 μmol, 1.0 equiv) in anhydrous DCM (500 μL) was added B (32.2 mg, 24.2 μmol, 3.8 equiv) and EtN (3.8 mg, 5.4 μL, 38 μmol, 6.0 equiv). After stirring at ambient temperature for 6 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% to 15% MeOH in DCM) to give H as a solid (5.1 mg, 2.8 μmol, 43%). 84 H 108 N 13 O 28 S + (M+H +LCMS (ESI+) calculated for 1779.89, found 1780.40.

[0236] Example 6: Synthesis of Compound L [ka]

[0242] To a solution of Alloc-PBD derivative I (33.0 mg, 18.0 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (600 μL, degassed with N) was added pyrrolidine (3.9 mg, 4.6 μL, 55.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (54.0 μL, 16.8 mM, 0.91 μmol, 0.05 equiv.). After 30 min at ambient temperature, the reaction mixture was diluted with DCM (4.0 mL), and hydromatrix (215 mg) was added. The mixture was concentrated in vacuo to give a free-flowing powder (coated hydromatrix). The residue was then purified by flash column chromatography on silica gel (0% → 100% MeOH in DCM) to give J as a white solid (9.8 mg, 5.7 μmol, 31%). 88 H 106 N 13 O 24 + (M+H + LCMS (ESI+) calculated for 1729.86, found 1730.58.

[0237]

[0243] To a solution of J (9.8 mg, 6.4 μmol, 1.0 equiv) in anhydrous DCM (500 μL) was added B (30.0 mg, 23.7 μmol, 4.2 equiv) and EtN (3.4 mg, 4.8 μL, 34 μmol, 6.0 equiv). After stirring at ambient temperature for 48 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% → 40% MeOH in DCM) to give K as a solid (4.6 mg, 1.7 μmol, 30%). 104 H 128 N 15 O 31 S + (M / 2+H +LCMS (ESI+) calculated for 1058.14, found 1058.86.

[0238]

[0244] To a solution of K (4.6 mg, 1.7 μmol, 1.0 equiv.) in DMF (250 μL) was added EtN (4.2 mg, 5.8 μL, 42 μmol, 25.0 equiv.). After stirring at ambient temperature for 47 h, the reaction mixture was diluted to 900 μL with additional DMF and purified by preparative HPLC (Xbridge prep C column). 18 The product was purified by chromatography on a 5 μm OBD, 30 × 150 mm tube, 30% → 95% MeCN in water (0.01% formic acid), (tube pre-filled with 300 μL 5% aqueous NH4HCO3). The product-containing fractions were combined and concentrated in vacuo to give L as a solid (3.4 mg, 1.8 μmol). 90 H 118 N 15 O 31 S + (M+H + LCMS (ESI+) calculated for 1938.05, found 1939.56.

[0239] Example 7: Synthesis of Compound O [ka]

[0245] To a solution of Alloc-PBD derivative M (26.0 mg, 19.0 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (1.1 mL, degassed with N) was added pyrrolidine (4.1 mg, 4.8 μL, 58.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (80.0 μL, 16.8 mM, 1.39 μmol, 0.07 equiv.). After 105 h at ambient temperature, the reaction mixture was diluted with DCM (4.0 mL) and directly purified by flash column chromatography on silica gel (0%→25% MeOH in DCM) to give N as a white solid (23.3 mg, 18.4 μmol, 96%). 61 H 83 N8O 17 S2 + (M+H +LCMS (ESI+) calculated for 1264.49, found 1264.10.

[0240]

[0246] To a solution of N (23.3 mg, 18.4 μmol, 1.0 equiv) in anhydrous DCM (400 μL) was added B (37.7 mg, 44.2 μmol, 2.4 equiv) and EtN (11.2 mg, 15.4 μL, 110.6 μmol, 6.0 equiv). After stirring at ambient temperature for 3 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% → 20% MeOH in DCM) to give O as a solid (9.2 mg, 5.4 μmol, 29%). 77 H 105 N 10 O 24 S3 + (M+H + LCMS (ESI+) calculated for 1650.91, found 1650.19.

[0241] Example 8: Synthesis of Compound R [ka]

[0247] To a solution of Alloc-PBD derivative P (12.0 mg, 7.7 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (500 μL, degassed with N) was added pyrrolidine (1.6 mg, 1.9 μL, 23.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (112.0 μL, 5.5 mM, 0.61 μmol, 0.08 equiv.). After 120 h at ambient temperature, the reaction mixture was diluted with DCM (4.0 mL) and directly purified by flash column chromatography on silica gel (0% → 25% MeOH in DCM) to give Q as a solid (7.5 mg, 5.1 μmol, 66%). 74 H 96 N 11 O 21 + (M+H + LCMS (ESI+) calculated for 1475.62, found 1475.50.

[0242]

[0248] To a solution of Q (7.5 mg, 5.1 μmol, 1.0 equiv) in anhydrous DCM (400 μL) was added B (23.0 mg, 17.8 μmol, 3.5 equiv) and EtN (3.0 mg, 4.2 μL, 30 μmol, 6.0 equiv). After stirring at ambient temperature for 4 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% to 20% MeOH in DCM) to give R as a solid (5.4 mg, 2.7 μmol, 53%). 90 H 118 N 13 O 28 S + (M+H + LCMS (ESI+) calculated for 1862.04, found 1862.35.

[0243] Example 9: Synthesis of Compound V [ka] [ka]

[0249] To a solution of Alloc-PBD derivative S (19.0 mg, 9.2 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (1 mL, degassed with N) and DMF (100 μL) was added pyrrolidine (2.0 mg, 2.3 μL, 27.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (303.0 μL, 4.5 mM, 1.36 μmol, 0.15 equiv.). After 150 min at ambient temperature, the reaction mixture was diluted with DCM (4.0 mL) and hydromatrix was added. The mixture was concentrated in vacuo to give a free-flowing powder (coated hydromatrix). The residue was then purified by flash column chromatography on silica gel (0% → 80% MeOH in DCM) to give T as a solid (10.0 mg, 5.0 μmol, 55%). 107 H 123 N 12 O 26 + (M+H +LCMS (ESI+) calculated for 1993.19, found 1993.67.

[0244]

[0250] To a solution of T (10.0 mg, 5.0 μmol, 1.0 equiv) in anhydrous DCM (600 μL) and DMF (200 μL) was added B (15.0 mg, 20.0 μmol, 4.0 equiv) and EtN (1.5 mg, 2.1 μL, 15 μmol, 3.0 equiv). After stirring at ambient temperature for 18 h, the reaction mixture was directly purified by flash column chromatography on silica gel (0% → 25% MeOH in DCM) to give U as a solid (6.4 mg, 2.2 μmol, 43%). 123 H 145 N 14 O 33 S + (M / 2+H + LCMS (ESI+) calculated for 1190.3, found 1190.01.

[0245]

[0251] To a solution of T (6.4 mg, 2.2 μmol, 1.0 equiv.) in DMF (200 μL) was added EtN (3.3 mg, 4.5 μL, 32 μmol, 15.0 equiv.). After stirring at ambient temperature for 20 h, the reaction mixture was diluted to 900 μL with additional DMF and purified by preparative HPLC (Xbridge prep C column). 18 Purification was performed by HPLC using a 5 μm OBD, 30 × 150 mm tube, 30% → 95% MeCN in water (0.01% formic acid), (tube pre-filled with 300 μL 5% aqueous NH4HCO3). The product-containing fractions were combined and concentrated in vacuo to give V as a solid (1.9 mg, 0.94 μmol, 44%). 95 H 125 N 14 O 33 S + (M / 2+H + LCMS (ESI+) calculated for 1012.07, found 1012.30.

[0246] Example 10: Synthesis of Compound B2 [ka]

[0252] To a cold solution of TBCN-OH (201.3 mg, 1.23 mmol, 1.0 equiv) in acetonitrile (10 mL) was added chlorosulfonyl isocyanate (112.7 μL, 183.2 mg, 1.29 mmol, 1.05 equiv). After stirring on ice for 10 min, EtN (515.0 μL, 374.3 mg, 3.69 mmol, 3.0 equiv) and amino-PEG-4-acid (490.6 mg, 1.85 mmol, 1.5 equiv) were added. The mixture was stirred for 3.5 h, and DCM (15.0 mL) and demineralized water (15.0 mL) were added, and the product was extracted into the aqueous layer. EtOAc (20.0 mL) was added to the aqueous layer, and the aqueous layer was acidified with 1.0 M HCl solution until a pH of 4.0 was reached. The two layers were separated, and the aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated to give B2 as a clear yellow oil (404.1 mg, 0.78 mmol, 63%). 22 H 37 N2O 10 S + (M+NH4 + LCMS (ESI+) calculated for 538.63, found 538.56.

[0247] Example 11: Synthesis of Compound Z [ka]

[0253] To a solution of Alloc-PBD derivative X (15.0 mg, 8.7 μmol, 1.0 equiv., commercially available from MedChemExpress) in anhydrous DCM (300 μL, degassed with N2) was added pyrrolidine (1.9 mg, 2.1 μL, 26.0 μmol, 3.0 equiv.) and Pd(PPh3)4 (34.0 μL, 7.78 mM, 0.26 μmol, 0.03 equiv.). After 90 min at ambient temperature, the reaction mixture was diluted with DCM (2.0 mL) and washed with aqueous NH4Cl (sat., 1.5 mL). The two layers were separated, and the aqueous layer was extracted with DCM (2 × 2 mL). The combined organic layers were dried (Na2SO4) and directly purified by flash column chromatography on silica gel (0% → 25% MeOH in DCM) to give compound Y as an off-white solid (11.7 mg, 7.14 μmol, 82%). 83 H 104 N 11 O 24 + (M+H + LCMS (ESI+) calculated for 1639.77, found 1639.16.

[0248]

[0254] To a solution of Y (11.7 mg, 7.14 μmol, 1.0 equiv) in anhydrous DCM (400 μL) was added B2 (5.45 mg, 7.85 μmol, 1.1 equiv) and EDC.HCl (1.51 mg, 7.85 μmol, 1.1 equiv). After stirring at ambient temperature for 15 min, the reaction mixture was directly purified by flash column chromatography on silica gel (0%→20% MeOH in DCM) to give Z as an oil (8.1 mg, 3.8 μmol, 53%). 105 H 138 N 13 O 33 S + (M / 2+H + LCMS (ESI+) calculated for 1071.67, found 1071.81.

[0249] [Preparation of antibody-drug conjugates] General Procedure for Mass Spectral Analysis of Monoclonal Antibodies

[0255] Prior to mass spectrometry analysis, IgG was treated with IdeS, which allows for the analysis of the Fc / 2 fragment. For the analysis of the Fc / 2 fragment, a solution of 20 μg (unmodified) IgG was incubated with IdeS / Fabricator™ (1.25 U / μL) in PBS pH 7.4 in a total volume of 10 μL for 1 hour at 37°C. After diluting the sample to 80 μL, electrospray ionization time-of-flight (ESI-TOF) analysis was performed on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0250] Example 12. Conjugation of azide-modified trastuzumab with compound E to obtain conjugate trast-E.

[0256] A bioconjugate according to the present invention was prepared by conjugation of Compound E as a linker-conjugate with Trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of Trastuzumab-(6-N3-GalNAc)2 (728 μL, 15.0 mg, 20.6 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (122 μL), DMF (110 μL), and Compound E (40 μL, 10 mM solution in DMF) were added. The reaction mixture was incubated overnight at room temperature and then purified on Superdex200 Increase 10 / 300 GL (GE Healthcare) in an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26127 Da, approximately 90% of all Fc / 2 fragments, calculated mass 26129 Da).

[0251] Example 13. Conjugation of azide-modified B12 with compound E to obtain conjugate B12-E.

[0257] A bioconjugate according to the present invention was prepared by conjugation of Compound E as a linker-conjugate with B12-(6-N3-GalNAc)2 as a biomolecule. To a solution of B12-(6-N3-GalNAc)2 (288 μL, 7.0 mg, 24.3 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (108 μL), DMF (51 μL), and Compound E (18.7 μL, 10 mM solution in DMF) were added. The reaction mixture was incubated overnight at room temperature and then purified on Superdex200 Increase 10 / 300 GL (GE Healthcare) in an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to conjugated Fc / 2 fragments (observed mass 26094 Da, approximately 70% of total Fc / 2 fragments, calculated mass 26097 Da) and one minor product corresponding to conjugated Fc / 2 fragments (observed mass 26223 Da, approximately 20% of total Fc / 2 fragments, calculated mass 26325 Da).

[0252] Example 14. Conjugation of azide-modified trastuzumab with compound G to obtain conjugate B12-G.

[0258] A bioconjugate according to the present invention was prepared by conjugation of compound G as a linker-conjugate with trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (2.951 mL, 70 mg, 23.72 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (1.016 mL), DMF (513 μL), and compound G (187 μL, 10 mM solution in DMF) were added. The reaction was incubated overnight at room temperature and then purified on Superdex200 Increase 16 / 600 GL (Cytiva) using AKTA Pure (Cytiva). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26143 Da, approximately 90% of all Fc / 2 fragments, calculated mass 26144 Da).

[0253] Example 15. Conjugation of azide-modified trastuzumab with compound L to obtain conjugate trastuzumab-L.

[0259] A bioconjugate according to the present invention was prepared by conjugation of compound L as a linker-conjugate with trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (2.974 mL, 71 mg, 23.72 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (1.064 mL), DMF (524 μL), and compound L (188 μL, 10 mM solution in DMF) were added. The reaction mixture was incubated overnight at room temperature and then purified on Superdex200 Increase 16 / 600 GL (Cytiva) using AKTA Pure (Cytiva). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26301 Da, approximately 90% of all Fc / 2 fragments, calculated mass 26302 Da).

[0254] Example 16. Conjugation of azide-modified trastuzumab with compound O to obtain conjugate trastuzumab-O.

[0260] A bioconjugate according to the present invention was prepared by conjugation of Compound O as a linker-conjugate with Trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of Trastuzumab-(6-N3-GalNAc)2 (4.344 mL, 100 mg, 23.02 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (1.323 mL), DMF (733 μL), and Compound O (267 μL, 10 mM solution in DMF) were added. The reaction mixture was incubated overnight at room temperature and then purified on Superdex200 Increase 26 / 600 GL (Cytiva) in AKTA Pure (Cytiva). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26015 Da, approximately 90% of all Fc / 2 fragments, calculated mass 26015 Da).

[0255] Example 17. Conjugation of azide-modified trastuzumab with compound R to obtain conjugate trastuzumab-R.

[0261] A bioconjugate according to the present invention was prepared by conjugation of Compound R as a linker-conjugate with trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (4.344 mL, 100 mg, 23.02 mg / mL in TBS pH 7.5) prepared according to WO 2016170186, TBS pH 7.5 (1.323 mL), DMF (733 μL), and Compound R (267 μL, 10 mM solution in DMF) were added. The reaction mixture was incubated overnight at room temperature and then purified on Superdex200 Increase 26 / 600 GL (Cytiva) using AKTA Pure (Cytiva). Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26226 Da, approximately 90% of all Fc / 2 fragments, calculated mass 26226 Da).

[0256] Example 18. Conjugation of azide-modified trastuzumab with compound V to obtain conjugate trastuzumab-V.

[0262] A bioconjugate according to the present invention was prepared by conjugation of Compound V as a linker-conjugate with trastuzumab-(6-N3-GalNAc)2 as a biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (10.5 μL, 250 μg, 23.72 mg / mL in TBS pH 7.5) prepared according to WO2016170186, TBS pH 7.5 (3.6 μL) and Compound V (2.7 μL, 10 mM solution in DMF) were added. The reaction was incubated overnight at room temperature. Mass spectral analysis of the Fabricator-digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (observed mass 26386 Da, approximately 80% of the total Fc / 2 fragments, calculated mass 26387 Da) and one minor product corresponding to the starting material (observed mass 24364 Da, approximately 10% of the total Fc / 2 fragments, calculated mass 24365 Da).

[0257] In vitro and in vivo evaluation Example 19: Stability of trast-E in mouse, rat, and cynomolgus monkey (cyno) serum

[0263] Mouse, rat, and cynomolgus monkey sera were incubated with Protein A Sepharose (1 mL Sepharose / mL serum, commercially available from Repligen) at 4°C for 1 hour to deplete serum IgG. The depleted sera were filtered using a 0.22 μm filter (Millipore), divided into aliquots, rapidly cooled, and stored at -20°C until further use (multiple freeze-thaw cycles were avoided). Trast-E was added to a final concentration of 0.1 mg / mL and incubated at 37°C. After 7 days, samples (0.5 mL) were collected and stored at -80°C until further analysis. For analysis, samples were incubated with Protein A Sepharose (20 μL Sepharose, commercially available from Repligen) at room temperature for 1 hour. The beads were then washed with PBS (3 × 1 mL) and then eluted with 0.1 M glycine-HCl pH 2.7 (0.4 mL). After elution, samples were immediately neutralized with 1.0 M Tris pH 8.0 (0.1 mL) and spin-filtered to a final volume of approximately 50 μL in PBS pH 7.4. Mass spectral analysis of the fabricator-digested samples for the three whole sera showed one major product corresponding to conjugated Fc / 2 (observed masses 26130, 26130, and 26129 Da for mouse, rat, and cynomolgus serum, respectively; in all cases, approximately 90% of the total Fc / 2 fragments, calculated mass 26129 Da).

[0258] Example 20. In vivo efficacy in the JIMT-1 model

[0264] Female NOD / SCID mice (5–8 weeks old at the start of the study, obtained from GemPharmatech Co. Ltd., China) were cultured at 5 × 10 in 0.1 ml of PBS for tumor development. 6 JIMT-1 human breast cancer cells were inoculated subcutaneously into the right anterior abdominal region. 3When tumors reached the tumor-specific tumor (T2) range, groups of eight mice were injected intravenously with either vehicle, trast-E (at 1 mg / kg and 3 mg / kg), or B12-E (at 3 mg / kg). In all cases, a single dose was administered on day 0. Tumor volumes and body weights were measured twice weekly after randomization (Figure 13). Antitumor efficacy of trast-E was observed at both 1 mg / kg and 3 mg / kg, but not with the non-targeting control ADC B12-E.

[0259] Example 21. In vivo safety in SD rats

[0265] Female Sprague Dawley rats (5-7 weeks old at the start of the study, obtained from Vital River Laboratories Research Models and Services) were treated with vehicle (PBS pH 7.4) or a single dose of the antibody-drug conjugate trast-E at 10, 20, and 30 mg / kg. Each dose level was administered to a single rat. After administration, animals were examined daily for morbidity and mortality for 3 weeks. During regular monitoring, animals were examined for any effects of treatment on behavior, such as mobility, food and water consumption, weight gain / loss, dullness of eyes / hair, and any other abnormalities. Only mild weight loss was observed in rats treated with 20 and 30 mg / kg trast-E, but no weight loss was observed in rats treated with 10 and 30 mg / kg trast-E (see Figure 14). No other adverse events were observed at any dose level during the 3-week observation period.

Claims

1. Conjugates according to structure (1): A.-[L-(D) ｘ ] ｙ （１） (In the ceremony AB is a cell binding agent; x is 1 or 2; y is 1 or 2; D is a masked PBD dimer payload; L is a linker connecting AB to D, where L is at least one -L ６ -Z １ - fragment, Z １ is a linking group containing the product of a cycloaddition reaction, and L ６ is -GlcNAc(Fuc) ｗ - (G) ｊ -S-(L ７ ) ｗ’ -, where L ６ is GlcNAc(Fuc) ｗ is connected to AB via: G is a monosaccharide, j is an integer ranging from 0 to 6; S is a sugar or sugar derivative; GlcNAc is N-acetylglucosamine; Fuc is fucose, w is 0 or 1; w′ is 0 or 1; L ７ is -N(H)C(O)CH ２ -, -N(H)C(O)CF ２ - or -CH ２ -).

2. When y=1 and x=1, L is according to structure (2), ６ is connected to AB, and L Ｍ is linked to D; When y=2, L is according to structure (3), ６ is connected to AB, and L Ｍ is connected to D: 【Chemistry 1】 BM is a branching moiety selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic or a polycyclic moiety; z' and c' are both independently 0 or 1; L Ｂ , L Ｃ and L Ｍ is a linker, and L Ｍ is the structure -(L １ ) ｎ - (L ２ ) ｏ - (L ３ ) ｐ - (L ４ ) ｑ - is a L １ , L ２ , L ３ and L ４ are Z, L respectively. Ｃ or a linker linking BM to D together; n, o, p, and q are each independently 0 or 1, provided that p+q=1 or 0 and o=p; Linker L １ When x=1: -(W) ｋ -(A) ｄ -(B) ｅ -(A) ｆ -(B) ｇ - (W) ｇ - is expressed as Or if x=2: -(W) ｋ -(A) ｄ -(B) ｅ -(A) ｆ -(C(O)) ｇ -BM [-(A) ｄ’ -(B) ｅ’ -(A) ｆ’ - (W) ｇ’ -] ２ is expressed as During the ceremony, d and d′ are independently 0 or 1; l is 0 or 1; e and e' are independently integers ranging from 1 to 10; f and f′ are independently 0 or 1; g and g' are independently integers ranging from 0 to 10; k=0 or 1, provided that if k=1, then d=0; Preferably, f+d=1 or 2; A is a sulfamide group represented by structure (L1a); 【Chemistry 2】 In the formula, a=0 or 1, and R １３ is hydrogen, C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (hetero)arylalkyl groups, １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (Hetero)arylalkyl groups include O, S and NR １４ and optionally substituted and optionally interrupted by one or more heteroatoms selected from R １４ are independently hydrogen and C １ ~C ４ alkyl groups; 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)(CH ２ ) ｍ C(O)NH- or -(4-Ph)CH ２ NHC(O)(CH ２ ) ｍ C(O)NH—, where m is an integer ranging from 0 to 10; B is L ２ , -(CH ２ ) ｘ —O— or —O—(CH ２ ) ｘ - individually selected from moieties, or (B) ｅ Ha-((CH ２ ) ｘ -O) ｅ１ - (CH ２ ) ｘ - moiety, where e1 is an integer ranging from 1 to 10, and each x is individually an integer ranging from 1 to 10; Linker L ２ is a peptide spacer; Linker L ３ is a para-aminobenzyloxycarbonyl (PABC) derivative according to structure (L3): 【Transformation 3】 During the ceremony Ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring; R ２１ is H, R ２６ or C(O)R ２６ and R ２６ is C １ ~C ２４ (hetero)alkyl group, C ３ ~C １０ (hetero)cycloalkyl group, C ２ ~C １０ (hetero)aryl group, C ３ ~C １０ Alkyl (hetero)aryl groups and C ３ ~C １０ (hetero)arylalkyl groups, which are O, S and NR ２８ and optionally substituted and optionally interrupted by one or more heteroatoms selected from R ２８ are independently hydrogen and C １ ~C ４ alkyl groups; Linker L ４ is a moiety according to structure (L4): 【Chemistry 4】 In the formula, A and R ２１ The conjugate of claim 1, wherein is as defined above.

3. The conjugate of claim 1 or 2, wherein D is according to any one of structures (D1) to (D4): 【Transformation 5】 During the ceremony, dx is an integer ranging from 1 to 10, preferably dx is 1 or 2, and most preferably dx is 1; 【Transformation 6】 is in the pyrrolidine ring or in the pyrrolidine ring and D ２ represents a single or double bond between D １ and D １ ' is H, OH or SO ２ X, where X is a halogen selected from fluorine, chlorine, bromine, or iodine; D ２ and D ２ ' are -H, -OH, and =CH ２ and -CH ３ Selected from: D ３ and D ３ ' are H, OH, OMe, SMe, and NMe, respectively. ２ , NO ２ , F, Cl, Br or I, preferably D ３ and D ３’ are both OMe; G and G' are each a single bond or CH ２ represents; J is a single bond, CH ２ , NH, -C≡C-, -C≡C-CH ２ represents - or O; and C １ and C ２ represents a capping group, preferably C １ and C ２ is the structure -L ４ -HM, -L ３ -L ２ -HM, (C1), (C2), or (C3) independently selected from: 【Transformation 7】 During the ceremony L ２ , L ３ and L ４ is as defined in claim 2, and L ３ or L ４ is linked to the nitrogen of D via a carbamate group, and in the case of (D3) and (D4) J=NH; HM is a hydrophilic moiety; C ２２ HM, NO ２ or H, more preferably C ２２ No ２ or H; C ２３ is H or C １ ~C ３ Alkyl chain, preferably C ２３ is H or methyl, more preferably C ２３ is H; R ２１ is as defined in claim 2; Q is selected from O, NH, NMe, or N-HM.

4. Z １ The conjugate of any one of claims 1 to 3, wherein the conjugate comprises a structure selected from structures (Z2) to (Z20) and (Z38a): 【Transformation 8】 (In the formula, L Ｍ or the connection to BM is represented by a wavy bond, B （－） is an anion and ring Z is selected from triazole, cyclohexene, dicyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, oxazoline, isoxazoline, pyrazoline or piperazine; Preferably Z １ is selected from structures (Z21) to (Z38a), more preferably Z １ is (Z29)). 【Chemistry 9】

5. L Ｍ The conjugate of any one of claims 1 to 4, wherein -D has a structure selected from the group consisting of (LM1) to (LM4): 【Chemistry 10】 (wherein the wavy line represents Z １ indicates the connection to L ２ is as defined in claim 2).

6. The conjugate of any one of claims 1 to 5, wherein D has the structure (D21): 【Chemistry 11】

7. Linker L Ｍ The conjugate of any one of claims 1 to 6, wherein p=1 when

8. where x=1 and y=2, and the conjugate has the structure AB-[LD] ２ The conjugate of any one of claims 1 to 7, having the formula:

9. The conjugate of any one of claims 1 to 8, wherein x=1 and y=1, and the conjugate has the structure AB-[L-D], where L has a structure selected from (L5) to (L7): 【Chemistry 12】

10. The conjugate of any one of claims 1 to 9, wherein the linker comprises a self-immolative linker that is cleaved by a mechanism different from that of the self-immolative linker of the cap.

11. The conjugate of any one of claims 1 to 10, wherein the conjugate has a structure selected from (4) to (7): 【Chemistry 13】 【Chemistry 14】

12. The conjugate has the structure (8): 【Chemistry 15】 and preferably has a linker L ２ is selected from Glu-Gly-Cit, Glu-Gly-Val, Ala-Asn, Asn-Ala, Pro-Leu-Gly or Asn-Asn.

13. 13. A pharmaceutical composition comprising 100 μg / kg to 10 mg / kg of the conjugate of any one of claims 1 to 12 and one or more pharmaceutically acceptable excipients.

14. A conjugate or pharmaceutical composition according to any one of claims 1 to 13 for use in medicine, preferably in cancer treatment.

15. A process for preparing the conjugate of any one of claims 1 to 12, comprising: i) A cell-binding agent comprising y core N-acetylglucosamine (GlcNAc) moieties is prepared by the method of formula S(F): ｘ -P (wherein, S(F １ ) ｘ is a reactive group Q １ x reactive groups F capable of reacting with １ and P is a nucleoside mono- or diphosphate) and a catalyst (the catalyst is a sugar derivative containing the compound S(F １ ) ｘ and contacting in the presence of a nucleotide sequence (which can transfer a moiety to said core-GlcNAc moiety) to obtain a modified antibody according to formula (26): AB-[(L ６ )-S{F １ } ｘ ] ｙ （２６） (In the ceremony AB is a cell binding agent; b is 0 or 1; L ６ is -GlcNAc(Fuc) ｗ - (G) ｊ -S-(L ７ ) ｗ’ -, G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L ７ is -N(H)C(O)CH ２ -, -N(H)C(O)CF ２ - or -CH ２ - and; F １ is a reactive moiety; x is 1 or 2; y is 1 or 2; ii) optionally reacting the modified antibody according to formula (26) with a compound of formula (27): 【Chemistry 16】 Obtaining a modified antibody represented by formula (28); 【Chemistry 17】 iii) reacting the modified antibody according to formula (26) or (28) with a compound selected from structures (9), (10) and (11), preferably via 1,3-dipolar cycloaddition, to obtain an antibody-conjugate according to structure (1); [Chemistry 18] wherein, if optional step ii) is performed, the compound used in step iii) is represented by structure (11), and, if optional step ii) is not performed, the compound used in step iii) is represented by structure (9) or (10).

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