Antibody-drug conjugates using two different types of topoisomerase I inhibitors

Abstract

An antibody-drug conjugate (ADC) having the formula AL, where A is an antibody or antibody fragment and L is a linker comprising, as a first payload, a cell membrane-permeable topoisomerase I inhibitor, preferably a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a cell membrane-permeable topoisomerase I inhibitor, preferably a cell membrane-permeable camptothecin cytotoxic molecule. Also described are pharmaceutical compositions comprising the ADC and at least one pharmaceutically acceptable ingredient. Also described are ADCs for use in methods of treating patients suffering from, at risk of developing, and / or diagnosed with a neoplastic disease.

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate (ADC) having the formula AL, wherein A is an antibody or antibody fragment, and L is a linker comprising, as a first payload, a cell membrane-permeable topoisomerase I inhibitor, preferably a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a topoisomerase I inhibitor, preferably a non-cell membrane-permeable camptothecin cytotoxic molecule. The present invention further relates to a pharmaceutical composition comprising the ADC and at least one pharmaceutically acceptable ingredient. Furthermore, the present invention relates to the ADC for use in a method of treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic disease. [Background technology]

[0002] DNA topoisomerase is an essential enzyme that stabilizes DNA supercoiling and disentangles DNA. Topoisomerase inhibitors have been widely used as anticancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap the TOP1 cleavage complex, camptothecin and its derivatives are promising anticancer drugs. To increase the accumulation of TOP1 inhibitors in cancer cells through tumor targeting, TOP1 inhibitor antibody-drug conjugates (TOP1-ADCs) have been developed and are commercially available. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypic anticancer ADCs (Han S. et al., 2022, Pharmaceuticals, 14, 1707).

[0003] One of the most prominent examples of a TOP1-ADC is DS-8201a, a HER2-targeted ADC with a novel DNA topoisomerase I inhibitor that has demonstrated impressive antitumor activity and a favorable safety profile in a broad selection of HER2-positive models (Ogitani et al. 2016, Clin Cancer Res; 22(20)), leading to its approval for several types of HER2-positive breast cancer, non-small cell lung cancer, and gastric / gastroesophageal junction adenocarcinoma (see also the package insert for Enhertu®). Surprisingly, DS-8201a showed better antitumor activity than other HER2-targeting ADCs (T-DM1 or trastuzumab emtansine), and it was shown that DS-8201a was effective even in patients who were refractory or resistant to trastuzumab emtansine (Andre F. et al., 2023, The Lancet, 401, 10390, pp. 1773-1785). Furthermore, DS-8201a, with a drug-to-antibody ratio (DAR) of approximately 8, remained effective against tumors with low HER2 levels, whereas T-DM1 (DAR of 3.5) had no effect on these tumors (Ogitani et al., 2016, Clin Cancer Res; 22(20)). The authors showed that DS-8201a enabled delivery of a larger cytotoxic payload to HER2-medium / low-expressing tumor cells than lower-DAR ADCs. Therefore, T-DM1 was not effective in these models, and the lower DAR of T-DM1 (DAR 3.5) compared with DS-8201a was considered to be one of the reasons. Furthermore, in another study (Ogitani et al., 2016, Cancer Sci, 107, 7, pp. 1039-1046), so-called "bystander lethality" was shown to be another distinction between DS-8201a and T-DM1. This is because solid tumors with heterogeneous target expression, i.e., solid tumors in which some tumor cells do not express tumor antigens and therefore cannot be reached by ADCs, are more difficult to treat than, for example, hematological malignancies.The authors confirmed that the DS-8201a payload, Dxd, was highly membrane-permeable, while the T-DM1, Lys-SMCC-DM1, had low levels of membrane permeability. For example, in vitro, DS-8201a killed both HER2-positive KPL-4 cells and HER2-negative MDA-MB-468 cells in coculture, whereas T-DM1 did not. In vivo evaluation was performed using an in vivo imaging system inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells. In vivo, DS-8201a reduced luciferase signal in mice, indicating suppression of the MDA-MB-468-Luc population; T-DM1 did not. Furthermore, DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated into the opposite foci of NCI-N87 tumors, suggesting that the bystander lethal effect of DS-8201a was observed only in cells adjacent to HER2-positive tumors, reducing concerns about systemic toxicity. These results demonstrate that DS-8201a has a potent bystander effect due to its highly membrane-permeable payload, making it useful for treating tumors with HER2 heterogeneity that are non-responsive to T-DM1.

[0004] In summary, the prior art provides the following for the development of a powerful and effective TOP1-ADC: A high drug-to-antibody ratio (DAR), ideally 8; and It is important to have a cytotoxic topoisomerase I inhibitor as a payload that is highly cell membrane permeable to exert bystander killing.

[0005] Although the prior art provides already efficient anti-cancer agents as outlined above, there is still a need to provide further means for the effective treatment of cancer, and more specifically, improvements are still needed, especially with regard to further improving the anti-tumor effects of anti-cancer agents. Summary of the Invention [Means for solving the problem]

[0006] The present invention is based at least in part on the surprising discovery that, contrary to the above prior art knowledge, unexpectedly, an ADC made using the same HER-2 targeting antibody as used in DS-8201a (=Enhertu®), which has a DAR of only 4 and contains two topoisomerase I inhibitors that are cell membrane permeable and two that are not, showed significantly improved antitumor efficacy compared to DS-8201a.

[0007] More specifically, as illustrated in more detail in the Examples section below, ADCs were generated that have two exatecans per antibody (an exemplary payload that exhibits cell membrane penetration) and two Gly-exatecans with glycine residues linked to the exatecans (an exemplary payload that does not exhibit cell membrane penetration).

[0008] Thus, the present invention is based at least in part on the surprising discovery that the combination of two different topoisomerase I inhibitors has a synergistic effect over the standard DS-8201a, primarily because Gly-exatecan can kill target-positive cells very efficiently (the payload accumulates intracellularly and does not diffuse), while, in addition, the cytotoxic payload with bystander activity can kill not only the targeted cancer cells but also neighboring cells that may not express the target, addressing tumor heterogeneity and reducing the risk of recurrence.

[0009] In view of the prior art and taking into account these beneficial and surprising effects, the technical problem underlying the present invention is the provision of anti-cancer agents with improved anti-tumor effects.

[0010] The technical problem is solved by providing the embodiments provided herein and characterized in the claims.

[0011] Therefore, the present invention provides A compound of the formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being As a first payload, a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and The present invention relates to antibody-drug conjugates (ADCs) having as a second payload a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable.

[0012] Thus, the present invention comprises three main components: antibody, a linker, and The present invention relates to an antibody drug conjugate (ADC) comprising a topoisomerase I inhibitor as a first and second payload, wherein the topoisomerase I inhibitor as the first payload is cell membrane permeable, and the topoisomerase I inhibitor as the second payload is not cell membrane permeable.

[0013] In certain embodiments, the present invention comprises three main components: antibody, a linker, and The present invention relates to an antibody drug conjugate (ADC) comprising a camptothecin cytotoxic molecule as a first and second payload, respectively, wherein the camptothecin cytotoxic molecule of the first payload is cell membrane-permeable, and the camptothecin cytotoxic molecule of the second payload is not cell membrane-permeable.

[0014] These three main components are described in more detail below.

[0015] The term "antibody-drug conjugate" (ADC) refers to a targeted therapy in which a cytotoxic drug (payload) is conjugated to an antibody specific for a particular antigen via a linker. The antibody directs the payload to cells expressing the target antigen, such as cancer cells, allowing for precise delivery of the cytotoxic agent to the desired cells, thereby minimizing damage to healthy tissue. In this application, antibody-drug conjugates are also referred to as "antibody-payload conjugates."

[0016] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE).

[0017] The antibody is preferably a monoclonal antibody. The antibody may be of human origin, but may also be derived from mouse, rat, goat, donkey, hamster, or rabbit. If the conjugate is for therapeutic purposes, the mouse or rabbit antibody may optionally be chimeric or humanized.

[0018] Antibodies can also be bispecific (e.g., DVD-IgG, crossMab, added IgG-HC fusions) or biparatopic. For an overview, see Brinkmann and Kontermann; Bispecific antibodies; Drug Discovery Today; 2015; 20(7); p. 838-47.

[0019] The term "antibody" also encompasses antigen-binding fragments of antibodies. The term "antibody fragment," as used herein, refers to a portion of an antibody molecule that retains the ability to specifically bind to an antigen. These fragments are derived from full-length antibodies and include, but are not limited to, Fab (fragment antigen-binding), F(ab')2, scFv (single-chain fragment variable), dsFv (disulfide-stabilized Fv), Fab', diabodies, nanobodies (VHH or single-domain antibodies), and domain antibodies (dAbs). Antibody fragments are engineered to retain antigen-binding function while being smaller and more versatile than full-length antibodies, making them particularly useful for therapeutic, diagnostic, and research applications due to their enhanced tissue distribution, reduced immunogenicity, and ease of production and manipulation.

[0020] Preferably, the linker according to the present invention, more preferably the peptide linker according to the present invention, is a C H The antibody or antibody fragment of the present invention is conjugated to glutamine residue 295 (Q295) in the C2 domain. H Preferably, it comprises two domains.

[0021] Fragments or recombinant variants of antibodies containing the CH2 domain can be, for example: Antibody formats containing only heavy chain domains (shark antibodies / IgNAR(VH-CH1-CH2-CH3-CH4-CH5)2 or camelid antibodies / hcIgG(VH-CH2-CH3)2) scFv-Fc(VH-VL-CH2-CH3)2 Fc fusion peptides containing an Fc domain and one or more receptor domains It could be.

[0022] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is an IgG antibody.

[0023] As used herein, "IgG" refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma gene. In humans, IgG includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, and IgG3. Full-length IgG consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing immunoglobulin domains VL and CL, and each heavy chain containing immunoglobulin domains VH, Cγ1 (Cγ2), and Cγ3 (Cγ4). H 1), Cγ2 (C H 2), and Oγ3 (C H In the context of human IgG1, according to the EU index as in Kabat, "C H "1" refers to positions 118 to 215, and C H 2 domain refers to positions 231-340 and C H The 3 domain refers to positions 341 to 447. IgG1 also contains a hinge domain, which in the case of IgG1 refers to positions 216 to 230.

[0024] In preferred embodiments, the antibody is an IgG1 or IgG4 antibody. In certain preferred embodiments, the antibody is a human IgG1 antibody.

[0025] More preferred embodiments of antibodies in view of the present invention are further outlined below.

[0026] In a particular preferred embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is an antibody comprising a mutation that reduces, ablates or abolishes FcγR binding.

[0027] Mutations in antibodies, preferably in the constant region of the antibody heavy chain, that reduce, ablate or eliminate FcγR binding are known in the art and are contemplated in the context of the present invention.

[0028] Without being bound by theory, it is known that the combination of the mutations Lue234Ala and Leu235Ala (according to Kabat position numbering) abolishes binding to FcγRI, IIa, and IIIa for both IgG1 and IgG4. This combination of mutations Lue234Ala and Leu235Ala in the constant region of an antibody heavy chain is commonly referred to as a "LALA mutation." This type of mutation is commonly known in the art as a modification in the Fc region of an antibody to reduce the effector function of the antibody. The article by Liu et al., Antibodies 9(4):64 (2020), which outlines the current technology of Fc engineering for modulating antibody effector function, particularly to improve the performance of antibodies in cancer therapy, also describes the above "LALA mutation."

[0029] Thus, in a preferred embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG antibody.

[0030] Thus, in a preferred embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG1 or IgG4 antibody.

[0031] More preferred embodiments of antibodies in accordance with the present invention that comprise an A at Kabat position 234 and / or an A at Kabat position 235 are further outlined below.

[0032] The linker, preferably a peptide linker, described in further more detail below, comprises a cytotoxic molecule, ie, a toxin, as the payload, which is a topoisomerase I inhibitor, preferably a camptothecin.

[0033] The terms "toxin" and "cytotoxic molecule" are known in the art and generally relate to any compound that is toxic to a cell or organism. A toxin / cytotoxic molecule may be produced by a cell or organism. However, a toxin / cytotoxic molecule may also be a chemical derivative or analog of a toxin produced by a cell or organism. In general, a toxin / cytotoxic molecule is understood to be, but is not limited to, a small molecule, peptide, or protein. Specific examples are neurotoxins, necrotizing toxins, hematotoxins, and cytotoxins.

[0034] According to the present invention, the toxin / cytotoxic molecule is a toxin / cytotoxic molecule used in the treatment of neoplastic diseases, i.e., the toxic / cytotoxic molecule is conjugated to an antibody that is delivered to or into the malignant cells due to the targeting specificity of the antibody.

[0035] In the present invention, the toxin / cytotoxic molecule is a topoisomerase I inhibitor, preferably camptothecin. A "topoisomerase I inhibitor," as used herein, refers to a molecule that functions as a topoisomerase I inhibitor and may include camptothecin and non-camptothecins. Examples of non-camptothecin topoisomerase I inhibitors are indolocarbazoles, dibenzonaphthyridines, and indenoisoquinolones. The term "camptothecin," as used herein, refers to camptothecin or a camptothecin derivative that functions as a topoisomerase I inhibitor. Exemplary camptothecins include, for example, topotecan, exatecan, deruxtecan, irinotecan, belotecan, rubitecan, gimatecan, ciratecan, cositecan, DX-8951f, SN38, BN 80915, lulotecan, AZ0132, CPT1, CPT2, Dxd(1), Dxd(2), 9-nitrocamptothecin, and aminocamptothecin. Various camptothecins, including camptothecin, have been described for use in treating human cancer patients. Some camptothecins are described, for example, in Kehrer et al., Anticancer Drugs, 12(2):89-105, (2001) or Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392). In certain embodiments, the camptothecin is an exatecan derivative shown as compound 10 in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392. In certain embodiments, the camptothecin derivative is glycinated exatecan (G-Exa; see, e.g., Figure 4).

[0036] In the present invention, camptothecin cytotoxic molecules are used as the first and second payloads, respectively, and the camptothecin cytotoxic molecule of the first payload is cell membrane-permeable, while the camptothecin cytotoxic molecule of the second payload is not cell membrane-permeable.

[0037] Methods for rendering a molecule (in the present case, a camptothecin cytotoxic molecule) cell membrane permeable or non-cell membrane permeable are known in the art.

[0038] Without being bound by theory, for example, as illustrated in the Examples section below, the camptothecin cytotoxic molecule of the second payload is rendered non-cell membrane permeable by covalently attaching a glycine residue to the primary amine on the F ring of the camptothecin cytotoxic molecule.

[0039] In a preferred embodiment, the glycinated camptothecin is a glycinated exacane having the following structure: [ka]

[0040] Those skilled in the art are aware of additional common strategies for reducing drug cell membrane permeability: small, uncharged lipophilic molecules are more likely to permeate across the cell barrier than their charged, hydrophilic counterparts; therefore, modifications of topoisomerase 1 inhibitors that increase their overall polarity and hydrophilicity (by adding charge via highly ionizable groups such as primary, secondary, or tertiary amines, carboxylates, phosphonates, sulfonates, nitros, etc.) will reduce cell membrane permeability.

[0041] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the second payload is modified to reduce its cell membrane permeability. The topoisomerase I inhibitors are preferably modified to reduce their cell membrane permeability by covalently linking the topoisomerase I inhibitor to a molecule that increases its overall polarity and / or hydrophilicity. In a preferred embodiment, the polarity and / or hydrophilicity of the topoisomerase I inhibitor can be increased by covalently linking the topoisomerase I inhibitor to an amino acid residue, preferably a glycine residue.

[0042] Furthermore, the ability of a molecule (in the present case, a topoisomerase I inhibitor conjugated to an antibody) to become cell membrane permeable or non-cell membrane permeable can be measured / determined by methods known in the art and as described herein. Without being bound by theory, this ability can be measured / determined as follows: The desired ADC can be incubated under co-culture conditions essentially as described by Ogitani et al. (Ogitani et al., 2016, Cancer Sci, 107, 7, pp. 1039-1046): antigen-positive and antigen-negative cells are co-cultured in vitro, and upon incubation with the ADC, cell viability is measured. If the ADC can preferentially kill target-positive cells, the payload does not penetrate adjacent antigen-negative cells ("no bystander activity"), whereas if the desired ADC kills both types of cells, the payload is cell membrane permeable ("bystander activity").

[0043] An ADC payload is considered "cell membrane permeable" within the meaning of the present invention if ADC incubation results in efficient killing of at least 70%, preferably 80%, more preferably 90%, and most preferably greater than 95% of target-positive and target-negative cells under said co-culture conditions.

[0044] An ADC payload is considered "non-cell membrane permeable" within the meaning of the present invention if ADC incubation results in efficient killing of at least less than 30%, preferably less than 20%, preferably less than 10%, and most preferably less than 5% of target-positive and target-negative cells under said co-culture conditions.

[0045] In certain embodiments, the first payload, i.e., the cell membrane permeable topoisomerase 1 inhibitor, is exatecan: [ka] is.

[0046] In such embodiments, exatecan is preferably attached to a self-immolative moiety contained in the linker via a primary amine on the F ring, allowing for release of the fully active, chemically unmodified payload.

[0047] The second payload is preferably a modified exatecan, in which the exatecan is linked via the primary amine on the F ring to a molecule that increases its overall polarity and / or hydrophilicity, preferably to an amino acid residue, more preferably to a glycine residue.

[0048] As mentioned, the present invention relates to an antibody-payload conjugate comprising an antibody conjugated to a linker.

[0049] A linker generally refers to a molecule that connects two or more parts of a conjugate or construct. Linkers are also often referred to as spacers.

[0050] It should be understood that the present invention is based on the surprising discovery that the combination of two different payloads, a cell membrane-permeable topoisomerase I inhibitor and a cell membrane-impermeable topoisomerase I inhibitor, results in potent antitumor activity. Thus, the present invention is not limited to a particular conjugation strategy, and any possible linker can be used in accordance with the present invention.

[0051] That is, linkers according to the present invention can be conjugated to antibodies using a variety of strategies, including nonspecific conjugation methods such as lysine conjugation, which involves NHS esters or isothiocyanates to react with amine groups on lysine residues, and cysteine ​​conjugation, which uses maleimides, iodoacetamides, or disulfide re-bridging reagents to target thiol groups of cysteine ​​residues. Additionally, site-specific conjugation strategies such as enzymatic conjugation (e.g., transglutaminase, sortase), unnatural amino acid incorporation, or click chemistry for precise and homogeneous ADCs can be utilized.

[0052] In a preferred embodiment, the linker is a peptide linker.

[0053] A "peptide linker" within the meaning of the present invention is a molecule comprising at least two amino acid residues, wherein the two amino acid residues are coupled via a peptide bond. The peptide linker can be modified with one or more reactive groups to allow the peptide linker to be conjugated to an antibody. For example, the peptide linker can be functionalized with maleimide to allow conjugation to a cysteine ​​residue of an antibody.

[0054] However, peptide linkers are expected to be suitable as substrates for microbial transglutaminase. In particular, peptide linkers are expected to be suitable for conjugation to glutamine residues contained in antibodies. To this end, peptide linkers according to the present invention must contain at least one amino acid residue containing a primary amine.

[0055] Thus, in certain embodiments, in a peptide linker according to the present invention, the primary amine contained in the amino acid residue is a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) a primary amine contained in the N-terminal amino acid residue having the structure NH2-(Y)-COOH is.

[0056] That is, in a preferred embodiment, the amino acid residue containing a primary amine is a lysine residue. In such an embodiment, the peptide linker comprises a peptide moiety that includes at least one lysine residue.

[0057] However, linkers according to the present invention may also include lysine mimetics or lysine derivatives, provided that the lysine mimetics or lysine derivatives contain a free primary amine in the amino acid side chain.

[0058] In certain embodiments, an amino acid residue containing a primary amine may be a lysine mimetic. The term "lysine mimetic," as used herein, refers to a compound that has a different structure from lysine but similar characteristics to lysine and can therefore be used to replace lysine in a peptide or protein without significantly altering the function and / or structure of the peptide or protein. In certain embodiments, a lysine mimetic may differ from lysine in the length or composition of the aliphatic chain connecting the primary amine and the α-carbon atom. Thus, in certain embodiments, a lysine mimetic may be ornithine, homolysine, or 2,7-diaminoheptanoic acid. In certain embodiments, a lysine mimetic may be a beta-amino acid, such as beta-homolysine.

[0059] In certain embodiments, the amino acid residue containing a primary amine may be a lysine derivative. The term "lysine derivative" as used herein refers to a lysine or lysine mimic, and one or more functional groups contained in the lysine or lysine mimic are modified or substituted. The amino group in the side chain of the lysine derivative is preferably unmodified so that it is available for conjugation to a glutamine residue in a protein. Therefore, the "lysine derivative" contained in the peptide linker according to the present invention preferably contains a modified or substituted α-amino and / or α-carboxyl group.

[0060] In certain embodiments, the primary amine contained in the amino acid residue may be the primary amine contained in the N-terminal amino acid residue having the structure NH2-(Y)-COOH.

[0061] In certain embodiments, the primary amine can be the α-amino group of an α-amino acid, which can be any proteinogenic α-amino acid, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

[0062] In certain preferred embodiments, the primary amine may be the α-amino group of a glycine residue, and in such embodiments, the glycine residue is preferably the N-terminal amino acid residue of the peptide linker, so that the α-amino group is available for conjugation to a glutamine residue via microbial transglutaminase.

[0063] An amino acid containing a primary amine may be a non-standard amino acid or a synthetic amino acid. As used herein, a "non-standard amino acid" may be any amino acid that is not part of the set of proteinogenic amino acids but can be obtained from natural sources. However, it should be noted that some non-standard amino acids can also be found in naturally occurring peptides and / or proteins. A "synthetic amino acid," as used herein, may be any molecule that falls within the general definition of an amino acid (NH2-(Y)-COOH), i.e., contains an amino group and a carboxyl group, but is not found in nature. Thus, non-natural amino acids are preferably obtained by chemical synthesis. It should be understood that the distinction between non-standard and synthetic amino acids may be indeterminate in some cases. For example, an amino acid defined as a synthetic amino acid may later be identified in nature and therefore reclassified as a non-standard amino acid. A non-standard or synthetic amino acid may be an α-, β-, γ-, δ-, or ε-amino acid.

[0064] In certain embodiments, an amino acid containing a primary amine may have the structure NH2-(Y)-COOH.

[0065] In certain embodiments, the moiety Y may comprise a carbon backbone of 1 to 200 atoms, optionally at least 10 atoms substituted with one or more atoms, e.g., 10 to 100 atoms or 20 to 100 atoms, optionally the carbon backbone is a straight chain hydrocarbon or comprises a cyclic group, a symmetrically or asymmetrically branched hydrocarbon, a monosaccharide, a disaccharide, a straight chain or branched oligosaccharide (asymmetrically or symmetrically branched), other naturally occurring straight chain or branched oligomer (asymmetrically or symmetrically branched), or more generally, any dimer, trimer, or higher oligomer (linear, asymmetrically or symmetrically branched) obtained from any chain growth or step growth polymerization process.

[0066] Y may further comprise any linear, branched and / or cyclic C 2~30 Alkyl, C 2~30 Alkenyl, C 2~30 Alkynyl, C 2~30 Heteroalkyl, C 2~30 Heteroalkenyl, C 2~30 It may be a heteroalkynyl, optionally interrupted by one or more homocyclic aromatic or heterocyclic groups; notably, any linear or branched C 2~5 Alkyl, C 5~10 Alkyl, C 11~20 Alkyl, -OC 1~5 Alkyl, -OC 5~10 Alkyl, -OC 11~20 Alkyl, or (CH2-CH2-O-) 1~24 or (CH2) x1 -(CH2-O-CH2) 1~24 -(CH2) x2 - group (wherein x1 and x2 are independently an integer selected from the range of 0 to 20), an amino acid, an oligopeptide, a glycan, a sulfate, a phosphate, or a carboxylate. 2~6 It may contain alkyl groups.

[0067] In certain embodiments, in a peptide linker according to the invention, Y is -(RC) nand n is an integer ranging from 1 to 20, 1 to 15, or 1 to 10.

[0068] That is, Y has the structure [ka] may have:

[0069] In certain embodiments, Y can be a substituted or unsubstituted alkyl or alkenyl chain. It should be understood that when Y is a substituted or unsubstituted alkenyl chain, there cannot be at least two R moieties attached to consecutive carbon molecules.

[0070] The term "substituted alkyl," as used herein, generally refers to an alkyl group having an additional group or groups bonded to any carbon of the alkyl group. That is, a substituted alkyl has the structure -(RC) n where each R can independently be hydrogen or a functional group such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups.

[0071] In certain embodiments, amino acids containing primary amines have the structure NH2-(Y)-COOH, where Y is -(RC) n - and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, n The at least one, two, three, four, or five moieties R contained therein can be functional groups such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups.

[0072] In certain embodiments, in a peptide linker according to the invention, at least one R moiety of each -(RC)- monomer is hydrogen.

[0073] That is, in certain embodiments, one R moiety of each -(RC)- monomer can be hydrogen, while the other R moiety can be a functional group such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups. Alternatively, one R moiety of each -(RC)- monomer can be hydrogen, and the other R moiety can be absent (in the case of an alkene). In certain embodiments, some -(RC)- monomers within a moiety Y can contain two hydrogen substituents, and some -(RC)- monomers within the same moiety Y can contain one hydrogen substituent and one substituent R as defined herein.

[0074] In certain embodiments, in a peptide linker according to the invention, both R moieties of each -(RC)- monomer are hydrogen.

[0075] In certain embodiments, the structure -(RC) n - may be an unsubstituted alkyl chain and has the structure -(RC) n All moieties R in - are hydrogen atoms. That is, in certain embodiments, the structure -(RC) n - can be a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group.

[0076] That is, in certain embodiments, amino acids containing primary amines have the structure NH2-(Y)-COOH, where Y is -(CH2) n-, where n is an integer from 1 to 20. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 15. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 10. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 9. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 8. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 7. In certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 6.

[0077] In certain embodiments, Y is the structure -(CH) n -wherein n is 1. That is, in certain embodiments, the amino acid containing a primary amine can be glycine.

[0078] In certain embodiments, Y is the structure -(CH) n -wherein n is 2. That is, in certain embodiments, the amino acid comprising a primary amine can be β-alanine.

[0079] In certain embodiments, Y is the structure -(CH) n -wherein n is 3. That is, in certain embodiments, the amino acid containing a primary amine can be 4-aminobutyric acid.

[0080] In certain embodiments, Y is the structure -(CH) n - (where n is 4). That is, in certain embodiments, the amino acid containing a primary amine can be 5-aminopentanoic acid. (An exemplary linker containing 5-aminopentanoic acid is shown in Figure 24.)

[0081] In certain embodiments, Y is the structure -(CH) n -wherein n is 5. That is, in certain embodiments, the amino acid containing a primary amine can be 6-aminohexanoic acid.

[0082] In certain embodiments, Y is the structure -(CH) n -wherein n is 6. That is, in certain embodiments, the amino acid containing a primary amine can be 7-aminoheptanoic acid.

[0083] In certain embodiments, Y is the structure -(CH) n -wherein n is 7. That is, in certain embodiments, the amino acid containing a primary amine can be 8-aminooctanoic acid.

[0084] In certain embodiments, Y is the structure -(CH) n -wherein n is 8. That is, in certain embodiments, the amino acid containing a primary amine can be 9-aminononanoic acid.

[0085] In certain embodiments, Y is the structure -(CH) n -wherein n is 9. That is, in certain embodiments, the amino acid containing a primary amine can be 10-aminodecanoic acid.

[0086] In certain embodiments, Y is the structure -(CH) n -wherein n is 10. That is, in certain embodiments, the amino acid containing a primary amine can be 11-aminoundecanoic acid.

[0087] In certain embodiments, the amino acid containing a primary amine has the structure NH2-(CH2) n -X-(CH2) n -COOH (wherein X is a substituted or unsubstituted alkyl or heteroalkyl chain, and n is an integer of 0-20, 0-10, or 0-6).

[0088] That is, in certain embodiments, an amino acid containing a primary amine has the structure NH2-(CH2) n It may have the formula -X-COOH, where X is a substituted or unsubstituted alkyl or heteroalkyl chain, and n is an integer from 1 to 20, 1 to 10, or 1 to 6.

[0089] In certain embodiments, the amino acid containing a primary amine has the structure NH2-(CH2) n -COOH, where X is a substituted or unsubstituted alkyl or heteroalkyl chain, and n is an integer from 1 to 20, 1 to 10, or 1 to 6.

[0090] In a preferred embodiment, the primary amine-containing amino acid comprises at least one methylene group (CH). More preferably, the at least one methylene group is directly coupled to the primary amine. That is, the primary amine-containing amino acid preferably comprises the structure NH-CH-.

[0091] In a preferred embodiment, the primary amine contained in the amino acid residue in the peptide linker according to the present invention is a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) Structure NH2-(CH2) n It is a primary amine contained in the N-terminal amino acid residue having —COOH (wherein n is an integer ranging from 1 to 10).

[0092] In certain embodiments, the payload is directly or indirectly attached to the N-terminus of the peptide linker. In such embodiments, the primary amine contained in the amino acid residue is preferably a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; more preferably, a primary amine in the side chain of a lysine residue.

[0093] In certain embodiments, in a peptide linker according to the invention, the linker comprises 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or fewer amino acid residues.

[0094] A peptide linker according to the present invention preferably comprises at least two amino acid residues and no more than 25 amino acid residues. In some embodiments, all amino acid residues included in a peptide linker according to the present invention form a single peptide. However, it should be understood that a peptide linker may comprise two or more peptide moieties. For example, in certain embodiments, a peptide linker may comprise two peptide moieties, where the two peptide moieties are covalently linked to each other rather than by a peptide bond. Examples of such peptide linkers will be provided further below.

[0095] In certain embodiments, in a peptide linker according to the invention, the net charge of the linker is neutral or positive.

[0096] The net charge of a peptide is generally calculated at neutral pH (7.0). In the simplest approach, the net charge is determined by adding the number of positively charged amino acid residues (Arg, Lys, and His) and the number of negatively charged ones (Asp and Glu), and calculating the difference between the two groups. If the linker contains a non-standard amino acid or amino acid derivative containing a charged functional group, one skilled in the art can calculate the net charge at neutral pH as appropriate.

[0097] In certain embodiments, the payload may also contribute to the net charge of the linker, however, those skilled in the art will be aware of methods to calculate the net charge of the entire linker, including any payload, preferably at neutral pH (7.0).

[0098] In certain embodiments, the net charge of a peptide linker is calculated solely based on the amino acid residues contained in the linker, including amino acid mimetics and amino acid derivatives. Thus, in certain embodiments, in a peptide linker according to the present invention, the net charge of the amino acid residues contained in the peptide linker is neutral or positive.

[0099] In certain embodiments, in a peptide linker according to the invention, the linker does not contain any negatively charged amino acid residues.

[0100] That is, the linker may not contain negatively charged amino acid residues, including negatively charged amino acid mimetics and amino acid derivatives. Negatively charged amino acid residues are amino acids, amino acid mimetics, or amino acid derivatives that have a negative charge at neutral pH (7.0). Negatively charged standard amino acids are glutamic acid and aspartic acid. However, negatively charged non-standard amino acids, amino acid mimetics, and amino acid derivatives are known in the art.

[0101] It should be noted that a peptide linker according to the present invention may contain one or more glutamic or aspartic acid residues, however, in such embodiments, it is preferred that the carboxyl group contained in the aspartic or glutamic acid side chain be coupled to the payload.

[0102] In certain embodiments, in a peptide linker according to the invention, the linker comprises at least one positively charged amino acid residue.

[0103] In certain embodiments, the peptide linker comprises a positively charged lysine residue that provides a primary amine for transglutaminase-mediated conjugation to the antibody. However, it is preferred herein that the peptide linker comprises at least one additional positively charged amino acid. The additional positively charged amino acid may be a standard amino acid residue such as arginine or histidine. However, the additional positively charged amino acid may also be a non-standard amino acid.

[0104] In certain embodiments, in a peptide linker according to the invention, the linker comprises at least one arginine residue.

[0105] It has been demonstrated herein that the linker containing arginine residue can be conjugated to glycosylated antibody with high efficiency.Therefore, it is preferred herein that the peptide linker according to the present invention comprises at least one arginine residue.It should be noted that arginine residue can also be replaced by arginine mimic or arginine derivative.

[0106] The arginine residue can be located at any position in the peptide linker. In certain embodiments, the arginine residue is adjacent to an amino acid residue containing a primary amine. In certain embodiments, the arginine residue is coupled to the N-terminus of an amino acid containing a primary amine, i.e., a lysine residue, a lysine mimetic, or a lysine derivative (e.g., an RK motif). In certain embodiments, the arginine residue is coupled to the C-terminus of an amino acid containing a primary amine, i.e., a lysine residue, a lysine mimetic, or a lysine derivative (e.g., a KR motif). In certain embodiments, the arginine residue is coupled to an amino acid containing a primary amine, i.e., a lysine residue, a lysine mimetic, or a lysine derivative, via another amino acid residue, preferably an alanine residue (KAR or RAK motif). In certain embodiments, the peptide linker comprises arginine and histidine residues.

[0107] In certain embodiments, in a peptide linker according to the invention, the linker comprises at least one histidine residue.

[0108] WO 2023 / 161291, the entirety of which is incorporated herein, demonstrates that linkers containing histidine residues can be conjugated to glycosylated antibodies with high efficiency. Therefore, it is preferred herein that the peptide linker according to the present invention contains at least one histidine residue. It should be noted that the histidine residue can also be replaced by a histidine mimic or histidine derivative.

[0109] The histidine residue can be located at any position in the peptide linker. In certain embodiments, the histidine residue is adjacent to an amino acid residue containing a primary amine. In certain embodiments, the histidine residue is coupled to the N-terminus of an amino acid containing a primary amine, i.e., a lysine residue, lysine mimetic, or lysine derivative (e.g., an HK motif). In certain embodiments, the histidine residue is coupled to the C-terminus of an amino acid containing a primary amine, i.e., a lysine residue, lysine mimetic, or lysine derivative (e.g., a KH motif). In certain embodiments, the histidine residue is coupled to an amino acid containing a primary amine, i.e., a lysine residue, lysine mimetic, or lysine derivative, via another amino acid residue, preferably an alanine residue (KAH or HAK motif). In certain embodiments, the peptide linker comprises histidine and arginine residues.

[0110] In a particular embodiment, in a peptide linker according to the invention, the linker comprises the sequence motif RK.

[0111] A peptide linker containing the sequence motif RK (arginyl-lysyl) can be conjugated to a glycosylated antibody with extremely high efficiency, even when the linker contains two or more payloads. It should be understood that the lysine residue contained in the RK motif contains a primary amine, through which the peptide linker is conjugated to the glutamine residue contained in the antibody. That is, the lysine residue contained in the RK motif is preferably an amino acid containing a primary amine.

[0112] It is preferred herein that motif RK consists of the amino acids arginine and lysine, however it should be understood that the arginine and / or lysine residues may be substituted with arginine mimetics / derivatives and / or lysine mimetics / derivatives.

[0113] That is, in certain embodiments, motif RK can include an arginine mimetic. As used herein, the term "arginine mimetic" refers to a compound that has a different structure from arginine but similar characteristics to arginine and can therefore be used to replace arginine in a peptide or protein without significantly altering the function and / or structure of the peptide or protein. An arginine mimetic can differ from arginine in the length or composition of the aliphatic chain connecting the guanidino group and the α-carbon atom. Alternatively, or in addition, an arginine mimetic can differ from arginine in the guanidino group itself. That is, an arginine mimetic can include a functional group with physicochemical properties similar to those of a guanidino group. In certain embodiments, an arginine mimetic can be homoarginine, 2-amino-3-guanidino-propionic acid, β-ureidoalanine, or citrulline.

[0114] In certain embodiments, motif RK may comprise an arginine derivative. The term "arginine derivative," as used herein, refers to an arginine or lysine mimetic, wherein one or more functional groups contained in the arginine or arginine mimetic are modified or substituted. The arginine derivative may be an arginine or arginine mimetic in which the guanidino group has been substituted or modified. In certain embodiments, the arginine derivative may be ω-methylarginine. In embodiments in which residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α-amino group has been modified or substituted. In certain embodiments, the α-amino group of the arginine or arginine mimetic may be acetylated.

[0115] In certain embodiments, motif RK may comprise a lysine mimetic or a lysine derivative as defined elsewhere herein.

[0116] In certain embodiments, motif RK may include lysine mimetics / derivatives and arginine mimetics / derivatives.

[0117] In certain embodiments, the lysine residue, or lysine mimetic or lysine derivative can be separated from the arginine residue, or arginine mimetic or arginine derivative by one amino acid residue. That is, the peptide linker of the present invention may comprise the sequence motif RXK or KXR, where X can be any amino acid. In a preferred embodiment, the lysine residue, or lysine mimetic or lysine derivative can be separated from the arginine residue, or arginine mimetic or arginine derivative by an alanine residue. That is, the peptide linker of the present invention can comprise the sequence motif RAK or KAR. WO 2023 / 161291, incorporated herein in its entirety, demonstrates that a linker containing the sequence motif KAR can be conjugated to a glycosylated antibody with extremely high conjugation efficiency.

[0118] In a particular embodiment, in a peptide linker according to the invention, the linker comprises the sequence motif HK.

[0119] A peptide linker containing the sequence motif HK (histidyl-lysyl) can be conjugated to a glycosylated antibody with extremely high efficiency, even when the linker contains two or more payloads. It should be understood that the lysine residue contained in the HK motif contains a primary amine, through which the peptide linker is conjugated to the glutamine residue contained in the antibody. That is, the lysine residue contained in the HK motif is preferably an amino acid containing a primary amine.

[0120] It is preferred herein that the motif HK consists of the amino acids histidine and lysine, however it should be understood that the histidine and / or lysine residues may be substituted with histidine mimetics / derivatives and / or lysine mimetics / derivatives.

[0121] That is, in certain embodiments, the motif HK can include a histidine mimetic. As used herein, the term "histidine mimetic" refers to a compound that has a different structure from histidine but similar characteristics to histidine and can therefore be used to replace histidine in a peptide or protein without significantly altering the function and / or structure of the peptide or protein. A histidine mimetic can differ from histidine in the length or composition of the aliphatic chain connecting the imidazole group and the α-carbon atom. Alternatively, or in addition, a histidine mimetic can differ from histidine in the imidazole group itself. That is, a histidine mimetic can include a functional group with similar physicochemical properties to the imidazole group. In certain embodiments, a histidine mimetic can be a homohistidine.

[0122] In certain embodiments, the motif HK may comprise a histidine derivative. The term "histidine derivative" as used herein refers to a histidine or histidine mimetic, wherein one or more functional groups contained in the histidine or histidine mimetic are modified or substituted. The histidine derivative may be a histidine or histidine mimetic in which the imidazole group is substituted or modified. In embodiments in which the residue H is located at the N-terminal position of the linker, the H may be a histidine derivative in which the α-amino group is modified or substituted. In certain embodiments, the α-amino group of the histidine or histidine mimetic may be acetylated.

[0123] In certain embodiments, the motif HK may comprise a lysine mimetic or a lysine derivative as defined elsewhere herein.

[0124] In certain embodiments, the motif HK may comprise a lysine mimic / derivative and a histidine mimic / derivative.

[0125] In certain embodiments, the lysine residue, or lysine mimetic or lysine derivative, can be separated from the arginine residue, or arginine mimetic or arginine derivative by one amino acid residue. That is, the peptide linker of the present invention can comprise the sequence motif HXK or KXH, where X can be any amino acid. In a preferred embodiment, the lysine residue, or lysine mimetic or lysine derivative, can be separated from the arginine residue, or arginine mimetic or arginine derivative by an alanine residue. That is, the peptide linker of the present invention can comprise the sequence motif HAK or KAH.

[0126] In a particular embodiment, in the peptide linker according to the present invention, the linker comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29 or 82 to 93.

[0127] That is, the peptide linker may contain any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29 or 82 to 93.

[0128] That is, in certain embodiments, the peptide linker can comprise the peptide sequence RKAA (SEQ ID NO: 1). Several linkers comprising the sequence RKAA are shown herein (see Figures 4, 6, and 7). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAA.

[0129] In certain embodiments, the peptide linker may comprise the peptide sequence RK. A linker comprising the sequence RK is shown herein (see Figure 8). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RK.

[0130] In certain embodiments, the peptide linker may comprise the peptide sequence ARK (SEQ ID NO: 2). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide ARK.

[0131] In certain embodiments, the peptide linker may comprise the peptide sequence RKARA (SEQ ID NO: 3). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKARA.

[0132] In certain embodiments, the peptide linker may comprise the peptide sequence RAAAA (SEQ ID NO: 4). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RAAAA.

[0133] In certain embodiments, the peptide linker may comprise the peptide sequence RKAAAAAA (SEQ ID NO: 5). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAAAAAA.

[0134] In certain embodiments, the peptide linker may comprise the peptide sequence RKAASGSG (SEQ ID NO: 6). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAASGSG.

[0135] In certain embodiments, the peptide linker may comprise the peptide sequence RKHA (SEQ ID NO: 7). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKHA.

[0136] In certain embodiments, the peptide linker may comprise the peptide sequence RKHAAA (SEQ ID NO: 8). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKHAAA.

[0137] In certain embodiments, the peptide linker may comprise the peptide sequence GGR (SEQ ID NO: 9). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide GGR.

[0138] In certain embodiments, the peptide linker may comprise the peptide sequence GGRG (SEQ ID NO: 10). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide GGRG.

[0139] In certain embodiments, the peptide linker may comprise the peptide sequence EARKAA (SEQ ID NO: 11). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide EARKAA. In addition, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker may also be considered as QARKAA (SEQ ID NO: 84).

[0140] In certain embodiments, the peptide linker may comprise the peptide sequence RKAEA (SEQ ID NO: 12). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAEA. In addition, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker may also be considered as RKAQA (SEQ ID NO: 85).

[0141] In certain embodiments, the peptide linker may comprise the peptide sequence HKA (SEQ ID NO: 13). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide HKA.

[0142] In certain embodiments, the peptide linker may comprise the peptide sequence RhKAA (SEQ ID NO: 14), where hK is homolysine. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RhKAA.

[0143] In certain embodiments, the peptide linker may comprise the peptide sequence XGRG (SEQ ID NO: 15), where X is the structure NH2-(CH2) n -COOH, where n is an integer from 1 to 20, preferably from 1 to 10. A linker comprising the sequence XGRG is exemplified in Figure 24. Preferably, one or more payloads are attached to the C-terminus of the peptide XGRG.

[0144] In certain embodiments, the peptide linker may comprise the peptide sequence RKVCit (SEQ ID NO: 16), where Cit is citrulline. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKVCit.

[0145] In certain embodiments, the peptide linker may comprise the peptide sequence RKAR (SEQ ID NO: 17). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAR.

[0146] In certain embodiments, the peptide linker may comprise the peptide sequence RKVA (SEQ ID NO: 18). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKVA.

[0147] In certain embodiments, the peptide linker may comprise the peptide sequence KAR (SEQ ID NO: 19). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide KAR.

[0148] In certain embodiments, the peptide linker may comprise the peptide sequence RKEAA (SEQ ID NO: 20). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKEAA. In addition, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker may also be considered as RKQAA (SEQ ID NO: 86).

[0149] In certain embodiments, the peptide linker may comprise the peptide sequence RKDA (SEQ ID NO: 82). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKDA. In addition, it is preferred that one or more payloads are attached to the side chain of an aspartic acid residue. It should be understood that when an amine containing payload is attached to the side chain of an aspartic acid residue, the peptide sequence of the linker may also be considered as RKNA (SEQ ID NO: 83).

[0150] In certain embodiments, the peptide linker may comprise the peptide sequence ERKAA (SEQ ID NO: 21). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide ERKAA. In addition, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker may also be considered as QRKAA (SEQ ID NO: 87).

[0151] In certain embodiments, the peptide linker may comprise the peptide sequence RKAH (SEQ ID NO: 22). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAH.

[0152] In certain embodiments, the peptide linker may comprise the peptide sequence RKAN (SEQ ID NO: 23). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAN.

[0153] In certain embodiments, the peptide linker may comprise the peptide sequence RKGGFG (SEQ ID NO: 24). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKGGFG.

[0154] In certain embodiments, the peptide linker may comprise the peptide sequence RKGP (SEQ ID NO: 25). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKGP.

[0155] In certain embodiments, the peptide linker may comprise the peptide sequence KRKAA (SEQ ID NO: 26). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide KRKAA. Additionally, it is preferred that one or more payloads are attached to one of the lysine residues, preferably to the side chain of the N-terminal lysine residue.

[0156] In certain embodiments, the peptide linker may comprise the peptide sequence SRKAA (SEQ ID NO: 27). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide SRKAA. Additionally, it is preferred that one or more payloads are attached to the side chain of a serine residue.

[0157] In certain embodiments, the peptide linker may comprise the peptide sequence DDRKAA (SEQ ID NO: 28). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide DDRKAA. In addition, it is preferred that one or more payloads are attached to the side chain of an aspartic acid residue. It should be understood that when an amine containing payload is attached to the side chain of an aspartic acid residue, the peptide sequence of the linker may also be considered as DNRKAA (SEQ ID NO: 88), NDRKAA (SEQ ID NO: 89), or NNRKAA (SEQ ID NO: 90).

[0158] In certain embodiments, the peptide linker may comprise the peptide sequence EERKValCit (SEQ ID NO: 29). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide EERKValCit. In addition, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker may also be considered as EQRKValCit (SEQ ID NO: 91), QERKValCit (SEQ ID NO: 92), or QQRKValCit (SEQ ID NO: 93).

[0159] In certain embodiments, the peptide linker is any one of the linkers shown in Figures 4, 6, 7 and 8.

[0160] In certain embodiments, a peptide linker according to the invention comprises 2 to 4 payloads.

[0161] In certain embodiments, the peptide linker according to the present invention comprises or contains two payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0162] In certain embodiments, the peptide linker according to the present invention comprises or contains three payloads: two topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0163] In certain embodiments, the peptide linker according to the present invention comprises or contains three payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and two topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0164] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and three topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0165] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: two topoisomerase I inhibitors that are cell membrane permeable as first payloads; and two topoisomerase I inhibitors that are not cell membrane permeable as second payloads.

[0166] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: three topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0167] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains two payloads: a first payload, a single camptothecin cytotoxic molecule that is cell membrane permeable; and a second payload, a single camptothecin cytotoxic molecule that is not cell membrane permeable.

[0168] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains three payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0169] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains three payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0170] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and three camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0171] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0172] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: three camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0173] The peptide linkers according to the present invention can be used to prepare antibody-payload conjugates with a payload-to-antibody ratio of 4 or greater using microbial transglutaminase. Naturally glycosylated antibodies have a single conjugation site at glutamine residue 295 (Q295) of the heavy chain. Because antibodies contain two heavy chains, conjugating a linker with two payloads to each of the glutamine residues results in an antibody-payload conjugate containing four payloads. Similarly, conjugating a linker with three or four payloads to each of the glutamine residues results in an antibody-payload conjugate containing six or eight payloads, respectively. Thus, in certain embodiments, the peptide linkers according to the present invention contain two, three, or four payloads.

[0174] The present inventors have demonstrated different methods for coupling two or more payloads to a peptide linker. In certain embodiments, two payloads can be coupled to the C-terminus of the peptide linker. In other embodiments, two payloads can be coupled to the N-terminus of the peptide linker. In yet other embodiments, one or two payloads can be coupled to the N-terminus and C-terminus of the peptide linker, respectively.

[0175] In certain embodiments, the peptide linker is the linker shown in FIG.

[0176] In embodiments in which the payload is not attached to the N-terminus or C-terminus of the peptide linker, it is preferred that each terminus is modified: the N-terminus of the peptide linker is preferably acetylated, and the C-terminus of the peptide linker is preferably amidated.

[0177] In addition to coupling a payload to the end of the peptide linker, one or more payloads can also be coupled to an amino acid side chain. Those skilled in the art will recognize that amino acid residues have functional groups on their amino acid side chains that allow for the coupling of payloads. Amino acids with functional groups on their side chains include, but are not limited to, those described by deGruiter et al. in Biochemistry 2017, 56, 30, 3863-3873. In addition, payloads can also be coupled to the side chains of non-standard amino acids, including, but not limited to, pAcF, CpK, pAMF, SCpHK, AzK, and Sec.

[0178] That is, in a specific embodiment, in the peptide linker according to the present invention, at least one payload is attached to the side chain of a glutamic acid, aspartic acid, tryptophan, cysteine, lysine, tyrosine, serine, or threonine residue contained in the peptide linker.

[0179] In certain embodiments, one or two payloads may be attached to the carboxylic acid of the glutamic acid or aspartic acid side chain.

[0180] In certain embodiments, one or two payloads may be attached to the amine of a lysine side chain.

[0181] In certain embodiments, one or two payloads may be attached to the thiol of the cysteine ​​side chain.

[0182] In certain embodiments, one or two payloads may be attached to the hydroxyls of serine, threonine, or tyrosine side chains.

[0183] The payload can be directly coupled to the peptide linker. For example, an amine-containing payload can be coupled to the C-terminus of the peptide linker via an isopeptide bond. Similarly, a carboxyl-containing payload can be coupled to the N-terminus of the peptide linker via an isopeptide bond, or a thiol-containing payload can be coupled to the side chain of a cysteine ​​residue contained in the peptide linker.

[0184] However, it is preferred herein that the payloads are coupled to the peptide linker via a chemical linker, particularly when the two payloads are to be attached to either the N- or C-terminus of the peptide linker, the use of a chemical linker between the two payloads and the N- or C-terminus is preferred.

[0185] Thus, in certain embodiments, at least one of the two or more payloads is attached to the peptide linker via a chemical linker.

[0186] Within the present invention, it is preferred that at least one of the two or more payloads is coupled to the peptide linker via a chemical linker, however, even more preferably, all payloads are coupled to the peptide linker via chemical linkers.

[0187] Chemical linkers can have various purposes. In certain embodiments, chemical linkers simply function as "adapters" for coupling one payload to a peptide linker. For example, a chemical linker containing an amine group can be used to couple a payload to the C-terminus of a peptide linker via an amide bond. In such embodiments, it is preferred that the chemical linker contains one or more functional groups other than amine, allowing the payload to be coupled to the chemical linker via these additional functional groups.

[0188] In certain embodiments, the chemical linker functions as an "amplifier moiety" for coupling several payloads to the peptide linker. For example, a chemical linker containing a disubstituted amine can be used as a dendron for linking two payloads. The disubstituted amine can function as a branching point, allowing for the attachment of multiple payload molecules, thereby increasing the drug-to-antibody ratio (DAR). The chemical linker containing a disubstituted amine can have the following structure: [payload]-NH-[payload] (wherein each [payload] is linked directly or indirectly to a nitrogen (N).

[0189] The disubstituted amine can be linked to a carboxyl group contained in the linker via an amide bond. Preferably, the disubstituted amine is linked to the C-terminus of the peptide linker or the N-terminus of the peptide linker via a dicarboxylic acid, as described elsewhere herein. An example of an amplifier containing a disubstituted amine is the N-(2-carboxyethyl)-alanine (CEA) moiety.

[0190] An example of such an amplifier is shown in FIG.

[0191] Another example of an amplifier is the 2,6-bis-(hydroxymethyl)-p-cresol moiety. For example, chemical linkers containing disubstituted carboxylic acids can be used as dendrons to connect two payloads.

[0192] Further strategies for coupling two or more payloads to a peptide linker are disclosed in WO 2023 / 161291, which is incorporated herein in its entirety.

[0193] Similarly, chemical linkers containing carboxyl groups can be used to couple one or more payloads to the N-terminus of a peptide linker via an amide bond. For example, a dicarboxylic acid molecule can be used to couple an amine-containing payload to the N-terminus of a peptide.

[0194] Additionally, chemical linkers containing compatible functional groups can be used to couple payloads to amino acid side chains contained in the peptide linker.

[0195] In any of the embodiments disclosed above, one of skill in the art can identify a suitable chemical linker for coupling a payload to a peptide linker, whether that chemical acts as an "adapter" or an "amplifier moiety." That is, one of skill in the art can identify a linker that has the functional groups required for coupling a payload of interest to functional groups contained in the peptide linker.

[0196] However, the chemical linker may serve other functions in addition to acting as an adapter between the payload and the peptide linker.

[0197] That is, in certain embodiments, the chemical linker is an enzymatically and / or chemically cleavable linker.

[0198] The cleavable linker may be any enzymatically and / or chemically cleavable linker known in the art, including but not limited to those described by Bargh et al. (Chem. Soc. Rev., 2019, 48, 4361), which is incorporated herein by reference in its entirety.

[0199] Cleavable linkers have the advantage that the release of the payload from the antibody can be controlled and / or accelerated. For example, one or more payloads can be coupled to the peptide linker via an enzymatically and / or chemically cleavable chemical linker.

[0200] In certain embodiments, the chemical linker is cleavable in vivo. Cleavable linkers may contain chemically or enzymatically labile or degradable bonds. Cleavable linkers generally rely on biological processes to release the payload, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific proteases or other enzymes inside or outside the cell. Cleavable linkers generally incorporate one or more chemical bonds that are chemically or enzymatically cleavable. In certain embodiments, the linker contains a chemically labile group such as a hydrazone and / or disulfide group. Linkers containing chemically labile groups take advantage of the different properties between plasma and some cytoplasmic compartments. The intracellular conditions that promote payload release for hydrazone-containing linkers are the acidic environment of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytosol, which contains high thiol concentrations, such as glutathione. In certain embodiments, the plasma stability of linkers containing chemically labile groups can be increased by introducing steric hindrance using substituents in the vicinity of the chemically labile group.

[0201] Acid-labile groups, such as hydrazones or carbonates, remain intact in the systemic circulation in the neutral pH environment of blood (pH 7.3-7.5) and undergo hydrolysis, releasing the payload, upon internalization of the ADC into the weakly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of cells. This pH-dependent release mechanism is associated with nonspecific release of the payload. To increase the stability of the hydrazone group of the linker, the linker may be altered by chemical modification, e.g., substitution, allowing for tuning to achieve more efficient release in lysosomes while minimizing loss during circulation. Hydrazone- or carbonate-containing linkers may contain additional cleavage sites, such as additional acid-labile and / or enzymatically labile cleavage sites.

[0202] Other acid-labile groups that can be included in chemical linkers include cis-aconityl-containing linkers. Cis-aconityl chemistry uses a carboxylic acid juxtaposed to the amide bond to accelerate amide hydrolysis under acidic conditions.

[0203] Cleavable chemical linkers may also contain disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release payloads upon internalization into cells, where the cytosol provides a significantly more reducing environment than the extracellular environment. Cleavage of disulfide bonds generally requires the presence of cytoplasmic thiol cofactors, such as (reduced) glutathione (GSH). As a result, disulfide-containing linkers are reasonably stable in the circulation and selectively release payloads in the cytosol. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to preferential cleavage of disulfide bonds inside cells. GSH has been reported to be present in cells at concentrations ranging from 0.5 to 10 mM, compared to concentrations of GSH or cysteine, the most abundant low-molecular-weight thiols in the circulation, which are significantly lower at approximately 5 μM. Tumor cells, where irregular blood flow leads to hypoxia, may experience enhanced activity of reductases and therefore even higher glutathione concentrations. In certain embodiments, the in vivo stability of disulfide-containing linkers can be enhanced by chemical modifications of the linker, for example, the use of steric hindrance adjacent to the disulfide bond.

[0204] Another type of cleavable linker that can be used is a chemical linker that is specifically cleaved by enzymes. Such linkers are usually peptide-based or contain a peptidic region that acts as a substrate for the enzyme. Peptide-based linkers tend to be more stable in plasma and extracellular environments than chemically unstable linkers. Peptide bonds generally have good serum stability because lysosomal protease activity is very low in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. Payload release from antibodies occurs specifically due to the action of lysosomal proteases, such as cathepsin, legumain, and plasmin. These lysosomal proteases can be present at high levels in certain tumor cells, but can also be found extracellularly in the tumor microenvironment. Peptide-based linkers can also be cleaved by non-lysosomal extracellular proteases, such as matrix metalloproteinases. Non-peptide-based linkers can also be specifically cleaved by glycosidases.

[0205] In exemplary embodiments, the cleavable peptide is a tetrapeptide such as Gly-Phe-Leu-Gly (SEQ ID NO: 30), Ala-Leu-Ala-Leu (SEQ ID NO: 31), Gly-Gly-Phe-Gly (SEQ ID NO: 32), or Ala-Ala, Ala-Arg, Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Va The linker may be selected from dipeptides such as 1, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, phenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Met-(D)Lys, and Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides due to the hydrophobicity of longer peptides. That is, a linker comprising an amino acid as set forth in SEQ ID NOs: 1-29 or 82-93 can further comprise any of the dipeptide or tetrapeptide motifs listed above. Preferably, the dipeptide or tetrapeptide motif listed above is coupled to the payload directly or via a self-immolative spacer. However, it should be understood that the peptide linker itself, such as any of the linkers comprising amino acids as set forth in SEQ ID NOs: 1-29 or 82-93, may be subject to enzymatic cleavage by endogenous peptidases or proteases.

[0206] The enzymatically cleavable linker may contain a self-immolative spacer to spatially separate the payload from the enzymatic cleavage site. Direct attachment of the payload to the peptide linker may result in proteolytic release of amino acid adducts of the payload, thereby impairing its activity. The use of a self-immolative spacer allows for removal of a fully active, unmodified payload upon amide or glycosidic bond hydrolysis.

[0207] In certain embodiments, the peptide linker according to the present invention is a) a p-aminobenzyl alcohol moiety; or b) a 2,4-bis(hydroxymethyl)aniline moiety; or c) p-aminobenzyl quaternary ammonium; or d) an ethylenediamine-based moiety; or e) an (aminomethyl)pyrrolidine-based moiety; or f) Aminomethyl moiety is a self-immolative linker comprising:

[0208] One self-immolative spacer is the bifunctional para-aminobenzyl alcohol group, which is linked to a peptide via its amino group to form an amide bond, while an amine-containing drug can be attached to the linker's benzyl hydroxyl group (PABC) via a carbamate functional group. The resulting prodrug is activated upon protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and the remainder of the linker group. Heterocyclic variants of this self-immolative group have also been described. See, for example, U.S. Pat. No. 7,989,434, incorporated herein by reference. The para-aminobenzyl alcohol moiety can also be used to link phenol- or hydroxyl-containing payloads via carbonate formation. The para-aminobenzyl moiety can also be used to link tertiary- or heteroaryl-amine-containing payloads via quaternary ammonium (PABQ) formation. That is, in certain embodiments, the quaternary ammonium cation contained in the p-aminobenzyl quaternary ammonium in a peptide linker according to the present invention is derived from an amine contained in the payload. Preferably, the amine contained in the payload is a tertiary amine or a heteroaryl-amine.

[0209] Another self-immolative spacer is the 2,4-bis(hydroxymethyl)aniline group, which is linked to peptides via the amino group to form an amide bond, while amine-containing drugs can be attached via two carbamate functionalities via the linker's two benzyl hydroxyl groups. The resulting prodrug is activated upon protease-mediated cleavage, resulting in payload release via sequential 1,6- and 1,4-elimination processes.

[0210] For hydroxyl-containing drugs, suitable self-immolative spacers include, but are not limited to, ethylenediamine-based carbamates (EDA), (aminomethyl)pyrrolidine-based carbamates (AMP) (see Figure 33), or aminomethyl moieties (AM). This latter release mechanism exploits the lability of the hemiaminal functional group, which readily undergoes 1,2-elimination to release the desired alcohol.

[0211] For thiol-containing drugs, suitable self-immolative spacers include, but are not limited to, aminomethyl moieties (AM). This latter release mechanism exploits the lability of the thiohemiaminal functional group, which readily undergoes 1,2-elimination to release the desired thiol.

[0212] In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. The easy release of the payload can be achieved through the cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and is overexpressed in some tumor types, while its extracellular enzymatic activity is low. β-glucuronic acid-based linkers can be used to avoid the tendency of antibody-payload conjugates to undergo aggregation due to the hydrophilic nature of β-glucuronides.

[0213] As noted above, in certain embodiments, the chemical linker is or comprises a self-immolative linker.

[0214] It is preferred herein that the payload is attached to the peptide linker via a self-immolative linker to facilitate the release of the unmodified drug. Even more preferably, the self-immolative linker is coupled to a peptide sequence that is efficiently cleaved by a protease or peptidase. The cleavable peptide can be defined as part of the peptide linker or as part of the chemical linker that connects the peptide linker to the payload.

[0215] The self-immolative linker can be any self-immolative linker known in the art, however, preferably the self-immolative linker comprises a p-aminobenzyl alcohol moiety or a 2,4-bis(hydroxymethyl)aniline moiety.

[0216] That is, in certain embodiments, the self-immolative linker comprises a p-aminobenzyl alcohol moiety or a 2,4-bis(hydroxymethyl)aniline moiety.

[0217] A self-immolative linker containing a p-aminobenzyl alcohol moiety can be used to couple a payload to the C-terminus of a peptide. That is, the amino group of the p-aminobenzyl alcohol moiety can be coupled to the C-terminal carboxyl group of the peptide linker via an amide bond. Alternatively, or in addition, the amino group of the p-aminobenzyl alcohol moiety can be coupled to the carboxyl group in the side chain of an aspartic acid or glutamic acid residue in the peptide linker via an amide bond.

[0218] The payload can be coupled to the hydroxyl group of the p-aminobenzyl alcohol moiety via a carbamate. In certain embodiments, the C-terminal amino acid of the peptide linker to which the p-aminobenzyl alcohol moiety can be coupled can be included in a motif that is efficiently cleaved by peptidases, such as, but not limited to, the sequence motif valine-citrulline.

[0219] It should be understood that the peptide linker according to the present invention can contain two or more p-aminobenzyl alcohol moieties.For example, the peptide linker according to the present invention can contain two peptide moieties, and the two peptide moieties are linked to each other via their N-termini.In such an embodiment, the peptide linker has two C-termini, and both C-termini can be conjugated to the payload via p-aminobenzyl alcohol moieties.

[0220] A p-aminobenzyl alcohol moiety can also be used to couple a payload to an amino acid side chain. For example, a payload can be coupled to the carboxyl group in the side chain of a glutamic acid or aspartic acid residue via the p-aminobenzyl alcohol moiety. The p-aminobenzyl alcohol moiety can be coupled to the carboxyl group in the side chain of a glutamic acid or aspartic acid residue directly or via one or more amino acid residues. In certain embodiments, the p-aminobenzyl alcohol moiety can be coupled to the carboxyl group in the side chain of a glutamic acid or aspartic acid residue via a valine-citrulline or alanine-alanine sequence.

[0221] In certain embodiments, the amine containing payload can be coupled to a carboxyl group in the peptide linker by two or more aminobenzyl alcohol moieties.

[0222] A self-immolative linker containing a 2,4-bis(hydroxymethyl)aniline moiety can be used to couple two payloads to a single functional group contained in a peptide linker. That is, the 2,4-bis(hydroxymethyl)aniline moiety can be coupled via its amino group to a carboxyl group contained in the peptide linker. Then, a payload can be coupled to each of the hydroxyl groups via a carbamate.

[0223] By using a linker containing a 2,4-bis(hydroxymethyl)aniline moiety, a peptide linker containing three or more payloads may be obtained. For example, a linker containing four payloads may be obtained by coupling two payloads to the N-terminal portion of the peptide linker via a 2,4-bis(hydroxymethyl)aniline moiety (indirectly via a second peptide moiety) and coupling two more payloads to the C-terminal portion of the peptide linker via another 2,4-bis(hydroxymethyl)aniline moiety. Similarly, a peptide linker containing three payloads may be obtained by coupling two payloads to the peptide linker via a 2,4-bis(hydroxymethyl)aniline moiety and a third payload via a p-aminobenzyl alcohol moiety.

[0224] In a particular embodiment, the present invention relates to a peptide linker according to the invention, wherein the hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbamate with the payload.

[0225] As described above, the payload can be linked to the p-aminobenzyl alcohol moiety via a carbamate. That is, the payload preferably contains a free amine group suitable for undergoing carbamate formation. Those skilled in the art will recognize methods for forming a carbamate between the p-aminobenzyl alcohol moiety and an amine comprising the payload.

[0226] In certain embodiments, in a peptide linker according to the invention, the hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbonate with the payload.

[0227] The payload can be linked to the p-aminobenzyl alcohol moiety via a carbonate. That is, the payload preferably contains a free hydroxyl group suitable for undergoing carbonate formation. Those skilled in the art will recognize methods for forming a carbonate between a p-aminobenzyl alcohol moiety and a hydroxyl-containing payload.

[0228] In certain embodiments, in a peptide linker according to the invention, each of the hydroxyl groups contained in the 2,4-bis(hydroxymethyl)aniline moiety forms a carbamate with the payload.

[0229] That is, the 2,4-bis(hydroxymethyl)aniline moiety contained in the peptide linker according to the present invention can form two carbamates with two individual amines comprising the payload.

[0230] In certain embodiments, in a peptide linker according to the invention, the p-aminobenzyl moiety forms a quaternary ammonium with the payload.

[0231] As mentioned above, the payload can be linked to p-aminobenzyl via a quaternary ammonium. That is, the payload preferably comprises a tertiary or heteroaryl-amine suitable for undergoing quaternary ammonium formation. Those skilled in the art will recognize methods for forming a quaternary ammonium between a p-aminobenzyl moiety and a payload comprising a tertiary or heteroaryl-amine.

[0232] In certain embodiments, in a peptide linker according to the invention, the self-immolative linker comprises an ethylenediamine carbamate (EDA) moiety.

[0233] That is, the payload can be coupled to the peptide linker via an ethylenediamine carbamate (EDA) moiety. The EDA moiety can be directly coupled to the C-terminus of a peptide or an aspartic acid or glutamic acid side chain via an amide bond. The EDA moiety preferably undergoes carbamate formation with a payload containing a hydroxyl group. The EDA moiety can also be used to link an amplifier linked to two payloads.

[0234] In certain embodiments, in a peptide linker according to the invention, the self-immolative linker comprises an (aminomethyl)pyrrolidine-based carbamate (AMP) moiety.

[0235] That is, a payload can be coupled to a peptide linker according to the present invention via an (aminomethyl)pyrrolidine-based carbamate (AMP) moiety. The AMP moiety can be coupled directly to the C-terminus of a peptide or an aspartic acid or glutamic acid side chain via an amide bond. The AMP moiety preferably undergoes carbamate formation with a payload containing a hydroxyl group.

[0236] In certain embodiments, in a peptide linker according to the invention, the self-immolative linker comprises an aminomethyl (AM) moiety.

[0237] That is, a payload can be coupled to the peptide linker of the present invention via an aminomethyl (AM) moiety. The AM moiety can be directly coupled to the C-terminus of a peptide or an aspartic acid or glutamic acid side chain via an amide bond. The AM moiety is preferably used to link a payload containing a hydroxyl group, thereby forming a hemiaminal. However, the AM moiety can also be used to link a payload containing a thiol group, thereby forming a thiohemiaminal.

[0238] In certain embodiments, in a peptide linker according to the present invention, at least one payload is attached to the side chain of a glutamic acid, aspartic acid, tryptophan, cysteine, lysine, tyrosine, serine, or threonine residue contained in the peptide linker.

[0239] As mentioned above, one or more payloads can be coupled to the amino acid side chains contained in the peptide linker.Those skilled in the art are aware of suitable chemical linkers for coupling payloads to amino acid side chains, i.e., carboxyl groups in the side chains of glutamic acid or aspartic acid residues, thiol groups in the side chains of cysteine ​​residues, amino groups in the side chains of lysine residues, or hydroxyl groups in the side chains of tyrosine, serine, or threonine residues.

[0240] In a particular embodiment, the present invention relates to a peptide linker according to the present invention, wherein the peptide linker comprises two peptide moieties, and the two peptide moieties are linked via their N-terminal amino acid residues with a dicarboxylic acid linker (HOC-R-COH).

[0241] Certain linkers within the scope of the present invention comprise two peptide moieties linked via their N-terminal amino acid residues, which may be linked via a dicarboxylic acid, each of whose carboxylic acid groups forms an amide bond with the N-terminal amino group of the peptide moiety.

[0242] Any dicarboxylic acid can be used to link two peptide moieties via their N-terminal amino acid residues. In certain embodiments, the dicarboxylic acid can be an aliphatic dicarboxylic acid. That is, the dicarboxylic acid is ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, or decanedioic acid. In certain embodiments, two peptide moieties are linked to a butanedioic acid molecule via their N-terminal amino acids. In certain embodiments, two peptide moieties are linked to a pentanedioic acid molecule via their N-terminal amino acids. The aliphatic dicarboxylic acid can include a substituted or unsubstituted alkyl or alkenyl chain.

[0243] In certain embodiments, the dicarboxylic acid can be an aromatic dicarboxylic acid, including, but not limited to, phthalic acid, isophthalic acid, or terephthalic acid.

[0244] It should be understood that linking two peptide moieties via their N-terminal amino acids results in a peptide construct that does not contain a free N-terminal amino group that may be suitable for conjugation to a glutamine residue contained in an antibody. Thus, a peptide linker comprising two N-terminally linked peptide moieties preferably contains a lysine residue, lysine mimetic, or lysine derivative that allows for conjugation of the peptide linker to a glutamine residue contained in an antibody.

[0245] In certain embodiments, the first peptide moiety included in the peptide linker may comprise any of the amino acid sequences set forth in SEQ ID NOS: 1-8, 11-14, 16-29, or 82-93. The second peptide moiety may have any amino acid sequence. In certain embodiments, the second peptide moiety may have a length of 2-100, preferably 2-50, more preferably 2-25, even more preferably 2-10, and most preferably 2-5 amino acid residues. In certain embodiments, the second peptide moiety may be a dipeptide or tripeptide. However, it should be noted that the second peptide moiety may also be a single amino acid or a longer peptide. To enable efficient release of the payload, the second peptide moiety preferably comprises a peptide sequence that is efficiently cleaved by peptidase. In certain embodiments, the second peptide portion may have the sequence Asn, Ala, Ala-Ala, Ala-Asn, Val-Ala, Val-Cit, Ala-Arg, Arg-Ala, Ala-Ala-Arg (SEQ ID NO: 34), Ala-Arg-Ala (SEQ ID NO: 35), Ala-Ala-Asn (SEQ ID NO: 36).

[0246] That is, in certain embodiments, the peptide linker has the structure: [payload 1]-[peptide 1]-[dicarboxylic acid]-[peptide 2]-[payload 2], wherein [Payload 1] and [Payload 2] are payloads, [Peptide 1] is the first peptide moiety, [Peptide 2] is a second peptide moiety, and [Dicarboxylic acid] is a dicarboxylic acid; at least one of peptide moieties 1 and / or 2 comprises a free amine; The N-terminus of peptide 1 and the N-terminus of peptide 2 are linked via a dicarboxylic acid, Payload 1 is preferably attached to the C-terminus of Peptide 1 via a chemical linker; and Payload 2 is preferably attached to the C-terminus of peptide 2 via a chemical linker.

[0247] Preferably, the peptide moiety comprising a free amine group is a peptide moiety comprising a lysine residue, a lysine mimetic or a lysine derivative as defined elsewhere herein, or a peptide linker comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1-8, 11-14, 16-29 or 82-93.

[0248] In certain embodiments, the peptide linker may comprise a first peptide portion comprising a sequence set forth in SEQ ID NOs: 1-8, 11-14, 16-29, or 82-93 and a second portion comprising the sequence Ala-Ala, wherein the first and second peptide portions are linked to a butanedioic acid molecule via their N-terminal amino acids.

[0249] In certain embodiments, the peptide linker may comprise a first peptide portion comprising the sequence RKAA and a second portion comprising the sequence Ala-Ala, wherein the first and second peptide portions are linked to the butanedioic acid molecule via their N-terminal amino acids.

[0250] Instead of coupling two peptide moieties via their N-terminal amino acid residues, the second peptide moiety can also be coupled to the amino acid side chain of the first peptide moiety. That is, the first peptide moiety included in the peptide linker can include any of the amino acid sequences set forth in SEQ ID NOs: 6, 11-12, 20-21, 23, 26-29, or 82-93. The second peptide moiety, which is disposed on the amino acid side chain, can have any amino acid sequence. In certain embodiments, the second peptide moiety can be a dipeptide or tripeptide. However, it should be noted that the second peptide moiety can also be a single amino acid or a longer peptide. To enable efficient release of the payload, the second peptide moiety preferably includes a peptide sequence that is efficiently cleaved by peptidase. In certain embodiments, the second peptide portion may have the sequence Asn, Ala, Ala-Ala, Ala-Asn, Val-Ala, Val-Cit, Ala-Arg, Arg-Ala, Ala-Ala-Arg (SEQ ID NO: 34), Ala-Arg-Ala (SEQ ID NO: 35), Ala-Ala-Asn (SEQ ID NO: 36).

[0251] The peptide linker according to the present invention comprises two or more payloads. The peptide linker comprising two or more payloads is preferably obtained by chemical synthesis.

[0252] Those skilled in the art are aware of methods for coupling a payload to an amino acid-based linker by chemical synthesis. For example, an amine-containing payload (e.g., for auristatin analogs, exatecan), a thiol-containing payload (e.g., for maytansine analogs), or a hydroxyl-containing payload (e.g., for SN-38 analogs) can be attached to the C-terminus of an amino acid-based linker by chemical synthesis. However, those skilled in the art are aware of additional reactions and reactive groups that can be used to couple a payload to the N-terminus, C-terminus, or side chain of an amino acid or amino acid derivative by chemical synthesis. Typical reactions that can be used to couple a payload to an amino acid-based linker by chemical synthesis include, but are not limited to, peptide coupling, activated ester coupling (NHS ester, PFP ester), click reaction (CuAAC, SPAAC), and Michael addition (thiol-maleimide conjugation).

[0253] Coupling of payloads to peptides has been reported, for example, by Costoplus et al. (Peptide-Cleavable Self-Immolative Maytansinoid Antibody-Drug Conjugates Designed to Provide Improved Bystander Killing. ACS Med Chem Lett. 2019 Sep 27;10(10):1393-1399), Sonzini et al. (Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry. Bioconjug Chem. 2020 Jan 15;31(1):123-129), Bodero et al. (Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting. Beilstein J. Org. Chem. 2018,14,407-415), and Nunes et al. (Use of a next-generation maleimide in combination with THIOMAB(TM) antibody technology delivers a highly stable, potent and near homogeneous THIOMAB(TM) antibody-drug conjugate(TDC).RSC Adv.,2017,7,24828-24832), Doronina et al.(Enhanced activity of monomethylauristatin F through monoclonal antibody delivery:effects of linker technology on efficacy and toxicity.Bioconjug Chem.2006 Jan-Feb;17(1):114-24), Nakada et al.(Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads. Bioorg Med Chem Lett. 2016 Mar 15;26(6):1542-1545) and Dickgiesser et al. (Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases. Bioconjug Chem. 2020 Mar 12. doi:10.1021 / acs.bioconjchem.0c00061) have been extensively described in the art.

[0254] It should be understood that the payload can be coupled to the N-terminus and / or C-terminus of the peptide-based or peptide-containing linker according to the invention. In certain embodiments, the payload can be coupled directly to the N-terminal amino group or C-terminal carboxyl group of the peptide or amino acid residue.

[0255] Those skilled in the art will recognize suitable reactive groups for coupling a payload to an amino acid residue.For example, a payload containing an amine can be coupled to the C-terminal carboxyl group of an amino acid residue via an amide bond.Alternatively, a payload containing a thiol group or / and a hydroxyl group can be coupled to the C-terminal carboxyl group of an amino acid via a thioester bond or an ester bond, respectively.A payload containing a carboxylic acid group can be coupled to the N-terminal amino group of an amino acid residue via an amide bond.

[0256] In certain embodiments, the payload may be indirectly coupled to the N-terminus and / or C-terminus of a peptide or amino acid residue contained in a linker according to the present invention. Those skilled in the art will recognize linker molecules that can be used to couple a payload to the N-terminal amino group or C-terminal carboxyl group of an amino acid residue contained in a linker according to the present invention.

[0257] In certain embodiments, a payload containing a hydroxyl group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing a hydroxyl group can be coupled to the N-terminal amino group via a carbamate linker.

[0258] In certain embodiments, a payload containing a thiol group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing a thiol group can be coupled to the N-terminal amino group via a thiocarbamate linker. Alternatively, a payload containing a thiol group can be coupled to the N-terminal amino group via an alkyl linker molecule containing a carboxyl group and a thiol group. In certain embodiments, the alkyl linker molecule can be a 3-mercaptopropionic acid linker molecule, and the payload forms a disulfur bond with the thiol group contained in the 3-mercaptopropionic acid linker molecule.

[0259] In certain embodiments, a payload containing an amide group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing an amine group can be coupled to the N-terminal amino group via a dicarboxylic acid linker molecule, with each carboxylic acid group contained in the dicarboxylic acid linker forming an amide bond with the payload and the amino group of the N-terminal amino acid residue. Examples of dicarboxylic acids that can be used as linker molecules in the present invention include, but are not limited to, succinic acid or pimelic acid.

[0260] Alternative linker molecules for indirectly coupling a payload to the N-terminus of an amino acid residue comprised in a peptide linker according to the invention or linker molecules suitable for indirectly coupling a payload to the C-terminus of an amino acid residue comprised in a peptide linker according to the invention have been described in the art and are encompassed by the present invention.

[0261] In another particular embodiment, one or more payloads are attached to the N-terminus of the amine-containing peptide linker and one or more payloads are attached to the C-terminus of said amine-containing peptide linker.

[0262] In certain embodiments, one or two payloads may be attached to the N-terminus of an amine-containing peptide linker, and one or two payloads may be attached to the C-terminus of the amine-containing peptide linker, i.e., the peptide linker may be a DAR4, DAR6, or DAR8 linker.

[0263] In certain embodiments, one payload may be attached to the N-terminus of an amine-containing peptide linker, and one payload may be attached to the C-terminus of the amine-containing peptide linker. In such embodiments, the peptide linker may be a "linear" DAR4 linker.

[0264] The amine-containing peptide linker can be any one of the lysine-containing peptide linkers disclosed herein, including peptide linkers that contain a lysine mimetic or lysine derivative as defined herein.

[0265] In certain embodiments, a "linear" DAR4 linker has the following structure (N->C orientation): [Payload 1]-[(Aa) m -(Lys)-(Aa) n ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa may be any amino acid residue; m and n may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; and Lys is a lysine residue, a lysine mimetic, or a lysine derivative; [Payload 1] is directly or indirectly attached to the N-terminus of the (Aa) or (Lys) residue, and [Payload 2] is directly or indirectly bound to the C-terminus of the (Aa) or (Lys) residue.

[0266] In certain embodiments, a "linear" DAR4 linker has the following structure: [Payload 1]-[(Aa) m -(Arg / His)-(Aa) n -(Lys)-(Aa) o ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa may be any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; and Arg may be an arginine residue, an arginine mimetic, or an arginine derivative; His may be a histidine residue, a histidine mimetic, or a histidine derivative; Lys is a lysine residue, a lysine mimetic, or a lysine derivative; [Payload 1] is attached directly or indirectly to the N-terminus of the (Aa) or (Arg / His) residue, and [Payload 2] is directly or indirectly bound to the C-terminus of the (Aa) or (Lys) residue.

[0267] In certain embodiments, a "linear" DAR4 linker has the following structure: [Payload 1]-[(Aa) m -(Lys)-(Aa) n -(Arg / His)-(Aa) o ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa may be any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; and Arg may be an arginine residue, an arginine mimetic, or an arginine derivative; His may be a histidine residue, a histidine mimetic, or a histidine derivative; Lys is a lysine residue, a lysine mimetic, or a lysine derivative; [Payload 1] is directly or indirectly attached to the N-terminus of the (Aa) or (Lys) residue, and [Payload 2] is directly or indirectly attached to the C-terminus of the (Aa) or (Arg / His) residue.

[0268] It should be understood that the payloads can be directly or indirectly attached to the N- and C-termini of the peptide linker. In one embodiment, a first payload can be directly attached to the N-terminal amino group of the peptide linker, and a second payload can be directly attached to the C-terminal carboxyl group of the peptide linker.

[0269] However, it is preferred that the payload be indirectly attached to the N-terminus and C-terminus of the peptide linker, for example, by any one of the chemical linkers described herein. In particular, the payload may be indirectly attached to the N-terminus of the peptide linker via a dicarboxylic acid and a second peptide moiety, as described in more detail elsewhere herein. Furthermore, it is preferred that all payloads be attached to the peptide or chemical linker via a self-immolative moiety, such as any one of the self-immolative moieties disclosed herein.

[0270] While the linker included in the ADCs according to the invention is preferably a peptide linker as described in detail herein, the linker may be any chemical linker, so long as the linker comprises at least a cell-membrane-permeable topoisomerase I inhibitor and a non-cell-membrane-permeable topoisomerase I inhibitor. Furthermore, the chemical linker may comprise any of the cleavable moieties described herein, particularly any of the self-immolative moieties.

[0271] As already explained above, the present invention relates to ADCs, i.e., antibody-linker conjugates comprising any of the linkers defined herein. That is, a linker comprising at least a cell membrane-permeable topoisomerase I inhibitor and a non-cell membrane-permeable topoisomerase I inhibitor can be conjugated to an antibody at any suitable conjugation site and by any suitable conjugation method.

[0272] As described herein above, the ADCs of the invention have the formula AL, where A is an antibody or antibody fragment, and L is a linker. Those skilled in the art will recognize that antibodies typically contain two identical light chains and two identical heavy chains. Consequently, any potential conjugation site in an antibody is typically present in both chains. Thus, those skilled in the art will understand that an ADC having the formula AL also encompasses ADCs having the formula LAL (or A-(L)2), in which one linker is conjugated to the corresponding position in each antibody chain.

[0273] Thus, in an alternative embodiment, the present invention provides a compound of formula LAL, wherein A is an antibody or antibody fragment and each L is a linker, said linker being: As a first payload, a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and The present invention relates to antibody-drug conjugates (ADCs) having as a second payload a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable. In such embodiments, the antibody is preferably a natural or engineered full-length antibody comprising two heavy chains and two light chains as described elsewhere herein, and even more preferably a full-length IgG antibody.

[0274] However, ADCs containing only a single conjugation site are also encompassed herein. Such ADCs may include, but are not limited to, single-chain antibody fragments or engineered antibodies containing only a single conjugation site. In such embodiments, the ADC may be conjugated with only a single linker and thus has the formula AL.

[0275] To encompass embodiments in which an antibody is conjugated to one or more linkers, an ADC may be defined to include the formula AL. Thus, in certain embodiments, the invention provides ADCs of the formula AL, where A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and The present invention relates to antibody-drug conjugates (ADCs) that contain, as a second payload, a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable.

[0276] It is preferred herein that the amine containing peptide linkers according to the present invention are conjugated to glutamine residues in the antibody. Such conjugation can be achieved by transglutaminase, as described in more detail elsewhere herein.

[0277] Thus, in certain embodiments, the invention relates to an ADC / antibody-payload conjugate according to the invention, wherein the peptide linker is conjugated to the antibody via an isopeptide bond formed between the γ-carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in an amino acid residue of the peptide linker.

[0278] Thus, one of skill in the art will readily understand that the primary amines contained in the peptide linkers disclosed herein are no longer present once the peptide linker is conjugated to an antibody.

[0279] The term "antibody" has already been defined above.

[0280] In certain embodiments, the invention relates to an ADC / antibody-payload conjugate according to the invention, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

[0281] That is, the peptide linker according to the present invention is preferably conjugated to a glutamine residue contained in the Fc domain of an antibody. The linker of the present invention can be conjugated to any Gln residue in the Fc domain of an antibody that can function as a substrate for transglutaminase. Typically, the term Fc domain, as used herein, refers to the last two constant region immunoglobulin domains (C) of IgA, IgD, and IgG. H 2 and C H 3) and the last three constant region domains of IgE, IgY, and IgM (C H 2. C H 3 and C H 4). That is, the linker according to the present invention is a C H 2. C H 3 and, where applicable, C H It can be conjugated to the 4 domain.

[0282] For example, a peptide linker according to the invention may be conjugated to an endogenous glutamine residue (eg, Q295 of an IgG1 antibody) or a glutamine residue that has been introduced into the Fc domain of an antibody by genetic engineering.

[0283] That is, in certain antibodies, the present invention relates to ADC / antibody-payload conjugates according to the invention, wherein the glutamine residue to which the peptide linker is conjugated is the C H The glutamine residue is Q295 (EU numbering) in the 2 domain.

[0284] It is important to understand that Q295 is a highly conserved amino acid residue in IgG type antibodies. It is particularly conserved in human IgG1, 2, 3, 4, as well as rabbit and rat antibodies. Therefore, being able to use Q295 is a considerable advantage for creating therapeutic antibody-payload conjugates. Although residue Q295 is highly conserved among IgG type antibodies, some IgG type antibodies, such as mouse and rat IgG2a antibodies, do not possess this residue. Therefore, the antibodies used in the methods of the present invention preferably contain a C H It should be understood that this is an IgG type antibody containing residue Q295 (EU numbering) in the 2 domain.

[0285] Transglutaminase-induced C H In the literature discussing the conjugation of linkers to two Gln residues, the focus has been on small, low-molecular-weight substrates. However, prior art literature describes the optional deglycosylation of the asparagine residue at position N297 or the use of aglycosylated antibodies to achieve such conjugation (WO 2015 / 015448; WO 2017 / 025179; WO 2013 / 092998).

[0286] However, quite surprisingly and contrary to all expectations, site-specific conjugation of glycosylated antibodies to Q295 was indeed possible using the peptide linker structures discussed above. In particular, the coupling of peptide linkers containing two or more payloads was achieved for the majority of them with conjugation efficiencies exceeding 90%.

[0287] Although Q295 is very close to N297, which is glycosylated in its native state, efficient conjugation to Q295 is still possible using the peptide linker specified.

[0288] Substitution of N297 with another amino acid may have undesirable effects, as it may affect the overall stability of the entire Fc domain (Subedi et al., The Structural Role of Antibody N-Glycosylation in Receptor Interactions. Structure 2015, 23(9), 1573-1583) and the efficacy of the entire conjugate, potentially resulting in increased antibody aggregation and decreased stability (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin 2011, 3(6), 568-576). Furthermore, the glycan present at N297 has important immunomodulatory effects, such as its ability to induce antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects would be lost upon deglycosylation or any of the other approaches discussed above to obtain aglycosylated antibodies. Furthermore, any sequence modification of an established antibody may also pose regulatory issues, which is problematic since very often accepted and clinically validated antibodies are used as the starting point for ADC conjugation.

[0289] Therefore, in view of the above, in the present invention, H For conjugation of IgG antibodies at residue Q295 (EU numbering) of the C2 domain, preferably H An antibody glycosylated at residue N297 (EU numbering) of domain 2 is used. However, the present invention also encompasses the conjugation of deglycosylated or non-glycosylated antibodies at residue Q295 or any other suitable Gln residue of an antibody, where it is explicitly stated that the Gln residue may be an endogenous Gln residue or a Gln residue introduced by molecular engineering.

[0290] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0291] The term "molecular engineering" as used herein refers to the use of molecular biology methods to manipulate nucleic acid sequences. In the context of the present invention, molecular engineering can be used to introduce Gln residues into the heavy or light chains of an antibody. Generally, two different strategies for introducing Gln residues into the heavy or light chains of an antibody are envisioned within the present invention. First, a single residue in the heavy or light chain of an antibody can be substituted with a Gln residue. Second, a Gln-containing peptide tag consisting of two or more amino acid residues can be incorporated into the heavy or light chain of an antibody. To this end, the peptide tag can be incorporated into an internal position of the heavy or light chain, i.e., between or by replacing two existing amino acid residues in the heavy or light chain, or the peptide tag can be fused (added) to the N-terminus or C-terminus of the heavy or light chain of an antibody.

[0292] For example, amino acid residues in the heavy or light chain of the antibody may be substituted with Gln residues, provided that the resulting antibody can be conjugated to a linker of the invention by microbial transglutaminase. In certain embodiments, the antibody has the C of an IgG antibody. H An antibody in which two amino acid residues N297 (EU numbering) are substituted, particularly an N297Q substitution. An antibody comprising an N297Q mutation can be conjugated to two or more linkers per heavy chain of the antibody. For example, an antibody comprising an N297Q mutation can be conjugated to four linkers, one linker conjugated to residue Q295 of the first heavy chain of the antibody, one linker conjugated to residue N297Q of the first heavy chain of the antibody, one linker conjugated to residue Q295 of the second heavy chain of the antibody, and one linker conjugated to residue N297Q of the second heavy chain of the antibody. Those skilled in the art will recognize that replacing residue N297 of an IgG antibody with a Gln residue results in a non-glycosylated antibody.

[0293] That is, in a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the glutamine residue introduced by molecular engineering into the heavy or light chain of the antibody is N297Q (EU numbering) in the CH2 domain of an aglycosylated IgG antibody.

[0294] In certain embodiments, the invention relates to an ADC / antibody-payload conjugate according to the invention, wherein the glutamine residue introduced into the antibody heavy or light chain by molecular engineering is either (a) incorporated into the antibody heavy or light chain or (b) contained in a peptide fused to the N- or C-terminus of the antibody heavy or light chain.

[0295] Instead of substituting a single amino acid residue in an antibody, a peptide tag containing a transglutaminase-accessible Gln residue can be introduced into the heavy or light chain of the antibody. Such a peptide tag can be fused to the N- or C-terminus of the heavy or light chain of the antibody. Alternatively, the peptide tag can be inserted into the heavy or light chain of the antibody at a suitable position. Preferably, a peptide tag containing a transglutaminase-accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, a peptide tag containing a transglutaminase-accessible Gln residue is fused to the C-terminus of the heavy chain of an IgG antibody. Several peptide tags that can be fused to the C-terminus of an antibody heavy chain and function as a substrate for microbial transglutaminase are described in WO 2012 / 059882 and WO 2016 / 144608.

[0296] Thus, in a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0297] Exemplary peptide tags that can be introduced into the heavy or light chain of an antibody, in particular, fused to the C-terminus of the heavy chain of an antibody, are LLQGG (SEQ ID NO: 70), LLQG (SEQ ID NO: 37), LSLSQG (SEQ ID NO: 38), GGGLLQGG (SEQ ID NO: 39), GLLQG (SEQ ID NO: 40), LLQ (SEQ ID NO: 41), GSPLAQSHGG (SEQ ID NO: 42), GLLQGGG (SEQ ID NO: 43), GLLQGG (SEQ ID NO: 44), GLLQ (SEQ ID NO: 45), LLQLLQGA (SEQ ID NO: 46), LLQGA (SEQ ID NO: 47), LLQYQGA (SEQ ID NO: 48), LLQGSG (SEQ ID NO: 49), LLQYQG (SEQ ID NO: 50), LLQLLQG ( SEQ ID NO: 51), SLLQG (SEQ ID NO: 52), LLQLQ (SEQ ID NO: 53), LLQLLQ (SEQ ID NO: 54), LLQGR (SEQ ID NO: 55), EEQYASTY (SEQ ID NO: 56), EEQYQSTY (SEQ ID NO: 57), EEQYNSTY (SEQ ID NO: 58), EEQYQS (SEQ ID NO: 59), EEQYQST (SEQ ID NO: 60), EQYQSTY (SEQ ID NO: 61), QYQS (SEQ ID NO: 62), QYQSTY (SEQ ID NO: 63), YRYRQ (SEQ ID NO: 64), DYALQ (SEQ ID NO: 65), FGLQRPY (SEQ ID NO: 66), EQKLISEEDL (SEQ ID NO: 67), LQR (SEQ ID NO: 68) and YQR (SEQ ID NO: 69).

[0298] Those skilled in the art know how to replace amino acid residues in an antibody or introduce peptide tags into an antibody by the method of molecular cloning as described, for example, in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0299] While it is preferred herein that the antibody be conjugated at a glutamine residue using transglutaminase as described hereinabove, the antibody may also be conjugated at a lysine residue. The lysine residue may be an endogenous lysine residue or a lysine residue that has been introduced into the antibody by genetic engineering. In such embodiments, the peptide linker may contain a glutamine residue that can form an isopeptide bond with a lysine residue of the antibody. The conjugation may be catalyzed by transglutaminase as described herein.

[0300] Generally, those skilled in the art know how to determine the position of an antibody to which a peptide linker is conjugated. For example, the conjugation site can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragments. For example, samples can be deglycosylated with GlyciNATOR (Genovis) according to the manufacturer's instructions, followed by digestion with trypsin gold (mass spectrometry grade, Promega). Thus, 1 μg of protein can be incubated with 50 ng of trypsin overnight at 37° C. LC-MS analysis can be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). To this end, 100 ng of peptide solution was loaded onto an Acquity PLC Symmetry C18 trap column (Waters, part no. 186006527) and captured with 1% Buffer A (water, 0.1% formic acid) and 99% Buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 5 μL / min for 3 min. Peptides were then eluted with a linear gradient from 3% to 65% Buffer B within 25 min. Data were acquired in positive polarity resolution mode and a mass range of 50–2000 m / z. Other instrument settings were as follows: capillary voltage 3.2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130 °C, cone gas 35 L / h, nanoflow gas 0.1 bar, and purge gas 150 L / h. The mass spectrometer was calibrated with [Glu1]-fibrinopeptide.

[0301] Additionally, those skilled in the art are aware of methods for determining the drug-to-antibody (DAR) or payload-to-antibody ratio of an antibody-payload construct. For example, the DAR can be determined by hydrophobic interaction chromatography (HIC) or LC-MS.

[0302] For hydrophobic interaction chromatography (HIC), samples may be adjusted to 0.5 M ammonium sulfate and run through a MAB PAK HIC butyl column (5 μm, 4.6 x 100 mm, Thermo Scientific) using a full gradient from A (1.5 M ammonium sulfate, 25 mM Tris HCl, pH 7.5) to B (20% isopropanol, 25 mM Tris HCl, pH 7.5) over 20 minutes at 1 mL / min and 30°C. Typically, 40 μg of sample may be used, and the signal may be recorded at 280 nm. The relative HIC retention time (HIC-RRT) may be calculated by dividing the absolute retention time of the two ADC DARs by the retention time of the respective unconjugated mAbs.

[0303] For LC-MS DAR determination, the ADC can be diluted with NH4HCO3 to a final concentration of 0.025 mg / mL. 40 μL of this solution can then be reduced with 1 μL of TCEP (500 mM) for 5 minutes at room temperature, followed by alkylation by adding 10 μL of chloroacetamide (200 mM), followed by incubation overnight at 37°C in the dark. For reversed-phase chromatography, a Dionex U3000 system combined with Chromeleon software can be used. This system can be equipped with a RP-1000 column (1000 Å, 5 μm, 1.0 × 100 mm, Sepax) heated to 70°C and a UV-detector set to a wavelength of 214 nm. Solvent A can consist of water with 0.1% formic acid, and solvent B can contain 85% acetonitrile with 0.1% formic acid. The reduced and alkylated sample may be loaded onto the column and separated by a gradient of 30-55% solvent B over a 14-minute period. The liquid chromatography system may be coupled to a Synapt-G2 mass spectrometer for identification of DAR species. The mass spectrometer capillary voltage may be set to 3 kV, the sampling cone to 30 V, and the extraction cone to 5 V. The source temperature may be set to 150 °C, the desolvation temperature to 500 °C, the cone gas to 20 L / h, and the desolvation gas to 600 L / h. Acquisition may be performed in positive mode over a mass range of 600-5000 Da with a 1 s scan time. The instrument may be calibrated with sodium iodide. Spectral deconvolution may be performed using the MassLynx MaxEnt1 algorithm until convergence. After assignment of DAR species to chromatographic peaks, the DAR may be calculated based on the integrated peak area of ​​the reversed-phase chromatogram.

[0304] In certain embodiments, the invention relates to an ADC / antibody-payload conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody.

[0305] That is, it is preferred herein that the peptide linker according to the present invention is conjugated to a glycosylated IgG antibody. It is particularly preferred that the peptide linker according to the present invention is conjugated to a naturally glycosylated IgG antibody. A naturally glycosylated IgG antibody contains a single conjugation site at glutamine residue 295 (Q295). Therefore, it is particularly preferred herein that the peptide linker according to the present invention is conjugated to residue Q295 of a naturally glycosylated antibody. The only glycosylation site of a naturally glycosylated IgG antibody is asparagine residue 297 (N297).

[0306] Thus, in certain embodiments, the invention relates to an ADC / antibody-payload conjugate according to the invention, wherein the IgG antibody is glycosylated at residue N297 (EU numbering) in the CH2 domain.

[0307] In a particularly preferred embodiment, the peptide linker according to the invention is conjugated to position Q295 of an IgG antibody that is glycosylated at position N297. More preferably, the antibody is an IgG1 antibody.

[0308] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker is a peptide linker.

[0309] In a preferred embodiment, the present invention relates to an ADC as defined herein, wherein the first payload and / or the second payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue comprised in the peptide linker; preferably The first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa.

[0310] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker has the following structure (N->C orientation): [Payload 1]-XZ-(Aa)mZ-(Aa) n(Lys)-(Aa)oZX-[Payload 2]; or [Payload 2]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z is absent or is a spacer comprising an alkyl or heteroalkyl group, preferably the spacer is (CH2)2; and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0311] More preferably, the invention relates to an ADC as defined herein, wherein the linker has the following structure: [Payload 1]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 2]; or [Payload 2]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2; and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0312] In a further preferred embodiment, residue (Aa) m +(Aa) n +(Aa) o is >0. Even more preferably, residue (Aa) n +(Aa) o is >0. That is, it is preferred herein that the Lys residue forms a peptide bound to at least one additional amino acid residue.

[0313] In a further preferred embodiment, the spacer Z2 is a dicarboxylic acid, as disclosed elsewhere herein, which connects the N-terminus of the peptide containing the (Lys) residue to the amino acid or peptide moiety (Aa) m By linking these two moieties via their N-termini, a peptide linker is formed that contains two C-termini. These two C-termini can be linked directly or indirectly to a payload, as disclosed elsewhere herein. In such embodiments, (Aa) m It should be understood that must be >0.

[0314] Thus, in certain embodiments, the invention relates to an ADC according to the invention, wherein Z2 is (Aa) m The N-terminus of (Aa) n or (Lys) is a dicarboxylic acid linked to the N-terminus of (Aa); and one payload is directly or indirectly linked to (Aa) m The other payload is directly or indirectly linked to the C-terminus of (Lys) or (Aa) o or vice versa.

[0315] In a more preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker has the following structure: [Payload 1]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[Payload 2]; or [Payload 2]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa)oX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer ranging from 1 to 10, preferably from 1 to 6, more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m The N-terminus of (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0316] As discussed hereinabove, the linker preferably comprises at least one positively charged amino acid residue in addition to the lysine residue. In certain preferred embodiments, the present invention relates to an ADC according to the invention, comprising (Aa) n -(Lys)-(Aa) o comprises the sequence motif Arg-Lys(RK) or His-Lys(HK) (N->C orientation). In another preferred embodiment, the invention relates to an ADC according to the invention, comprising (Aa) n -(Lys)-(Aa) ois or includes RK or RKAA (N->C direction).

[0317] In such embodiments, the moiety (Aa)m is preferably a single amino acid residue or a peptide having a length of 2 to 10, preferably 2 to 6, more preferably 2 to 4 amino acid residues. In certain embodiments, the moiety (Aa)m has or comprises the sequence Ala-Ala (AA) or Ala-Arg-Ala (ARA).

[0318] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0319] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker has the following structure: [ka] It comprises or consists of:

[0320] In a preferred embodiment, the present invention relates to an ADC as defined herein, said ADC comprising two or more first payloads and / or two or more second payloads.

[0321] As already outlined above, the peptide linker according to the invention comprises or contains at least two payloads.

[0322] When the peptide linker contains or comprises only two payloads, these two payloads are structurally different, i.e., the first payload is a camptothecin cytotoxic molecule that is cell membrane permeable; and the second payload is a camptothecin cytotoxic molecule that is not cell membrane permeable.

[0323] When a peptide linker comprises or contains three or more payloads, the payloads may be identical or different in structure, while at least two of the payloads may be identical in structure.

[0324] By coupling two or more identical payloads to a peptide linker, it becomes possible to increase the concentration of the payload in the target tissue or cell of the antibody-payload conjugate. For example, when the peptide linker of the antibody-payload conjugate contains two or more identical toxins (resulting in a DAR>4 ADC), the concentration of the toxin in the target tissue or cell is increased compared to a conventional DAR2 ADC. Using the peptide linkers of the present invention, ADCs containing 4, 6, or 8 identical payload molecules may be obtained.

[0325] In certain embodiments, a peptide linker according to the invention comprises 2 to 4 payloads.

[0326] In certain embodiments, the peptide linker according to the present invention comprises or contains two payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0327] In certain embodiments, the peptide linker according to the present invention comprises or contains three payloads: two topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0328] In certain embodiments, the peptide linker according to the present invention comprises or contains three payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and two topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0329] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and three topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0330] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: two topoisomerase I inhibitors that are cell membrane permeable as first payloads; and two topoisomerase I inhibitors that are not cell membrane permeable as second payloads.

[0331] In certain embodiments, the peptide linker according to the present invention comprises or contains four payloads: three topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0332] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains two payloads: a first payload, a single camptothecin cytotoxic molecule that is cell membrane permeable; and a second payload, a single camptothecin cytotoxic molecule that is not cell membrane permeable.

[0333] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains three payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0334] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains three payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0335] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and three camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0336] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0337] In a preferred specific embodiment, the peptide linker according to the present invention comprises or contains four payloads: three camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0338] The linker containing the three payloads has the following structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -Z3-X-[payload]; or [Payload]-X-Z1-(Aa) m -Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; (In the formula, [payload] is each independently a payload selected from a first and a second payload, and the linker comprises at least one of each of the first and second payloads; (Aa) is any amino acid residue; m, m*, n, o, p and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and even more preferably, n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxylic acid linker, and X may be absent or may carry a self-immolative group, preferably PABC.

[0339] More preferably, the linker comprising three payloads has the following structure: ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[payload]; or [Payload]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2; (In the formula, [payload] is each independently a payload selected from a first and a second payload, and the linker comprises at least one of each of the first and second payloads; (Aa) is any amino acid residue; m, m*, p, and p* are integers ranging from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa)m Or the N-terminus of the disubstituted amine (N) is (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and X may be absent or may carry a self-immolative group, preferably PABC.

[0340] The linker containing the four payloads has the following structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; (In the formula, [payload] is each independently a payload selected from a first and a second payload, and the linker comprises at least one of each of the first and second payloads; (Aa) is any amino acid residue; m, m*, n, o, p and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and even more preferably, n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxylic acid linker, and X may be absent or may carry a self-immolative group, preferably PABC.

[0341] More preferably, the linker comprising the four payloads has the following structure: ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o-N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2; (In the formula, [payload] is each independently a payload selected from a first and a second payload, and the linker comprises at least one of each of the first and second payloads; (Aa) is any amino acid residue; m, m*, p, and p* are integers ranging from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m Or the N-terminus of the disubstituted amine (N) is (Aa) n or (Lys); and X may be absent or may carry a self-immolative group, preferably PABC.

[0342] The linker preferably comprises, in addition to the lysine residue, at least one additional positively charged amino acid residue, as described in detail elsewhere herein.

[0343] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the camptothecin is exatecan or an exatecan derivative.

[0344] As already outlined above, the cytotoxic molecule of the ADCs of the invention is a topoisomerase I inhibitor, preferably a camptothecin. While numerous camptothecins are known in the art, including, for example, topotecan, exatecan, irinotecan, DX-8951f, SN38, BN80915, lutotecan, 9-nitrocamptothecin, and aminocamptothecin, the preferred camptothecin according to the invention is exatecan.

[0345] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein a second payload comprises an amino acid (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) residue linked to said topoisomerase I inhibitor (preferably said camptothecin cytotoxic molecule) of the second payload.

[0346] Without being bound by theory, the amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) has the function of making the second payload non-cell membrane permeable.

[0347] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein a second payload has an amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) linked to said topoisomerase I inhibitor of the second payload, thereby rendering it non-cell membrane permeable.

[0348] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload comprises an amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) as part of the linker connecting said topoisomerase I inhibitor of the second payload, thereby rendering it non-cell membrane permeable upon release of the corresponding amino acid-cytotoxic construct.

[0349] The ability of a molecule (in the present case, a topoisomerase I inhibitor) to become cell membrane permeable or non-cell membrane permeable can be measured / determined by methods known in the art and as described herein.

[0350] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload comprises a glycine residue linked to said topoisomerase I inhibitor (preferably, said camptothecin cytotoxic molecule) of the second payload.

[0351] Without being bound by theory, the glycine residue functions to make the second payload non-cell membrane permeable.

[0352] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload has a glycine residue linked to said topoisomerase I inhibitor of the second payload, thereby rendering it non-cell membrane permeable.

[0353] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload comprises a glycine residue as part of the linker connecting said topoisomerase I inhibitor of the second payload, thereby rendering it non-cell membrane permeable upon release of the glycine-cytotoxic construct.

[0354] The ability of a molecule (in the present case, a topoisomerase I inhibitor) to become cell membrane permeable or non-cell membrane permeable can be measured / determined by methods known in the art and as already described herein.

[0355] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein a second payload has an amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) linked to said camptothecin cytotoxic molecule of the second payload.

[0356] Without being bound by theory, the amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) has the function of making the second payload non-cell membrane permeable.

[0357] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein a second payload has an amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) linked to said camptothecin cytotoxic molecule of the second payload, thereby rendering it non-cell membrane permeable.

[0358] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload comprises an amino acid residue (preferably a glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue) as part of the linker that connects the second payload to said camptothecin cytotoxic molecule, thereby rendering it non-cell membrane permeable upon release of the corresponding amino acid-cytotoxic construct.

[0359] The ability of a molecule (in the present case, a camptothecin cytotoxic molecule) to become cell membrane permeable or non-cell membrane permeable can be measured / determined by methods known in the art and as described herein.

[0360] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload has a glycine residue linked to said camptothecin cytotoxic molecule of the second payload.

[0361] Without being bound by theory, the glycine residue functions to make the second payload non-cell membrane permeable.

[0362] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload has a glycine residue linked to said camptothecin cytotoxic molecule of the second payload, thereby rendering it non-cell membrane permeable.

[0363] Thus, in a preferred embodiment, the invention relates to an ADC as defined herein, wherein the second payload comprises a glycine residue as part of the linker that is linked to said camptothecin cytotoxic molecule of the second payload, thereby rendering it non-cell membrane permeable upon release of the glycine-cytotoxic construct.

[0364] The ability of a molecule (in the present case, a camptothecin cytotoxic molecule) to become cell membrane permeable or non-cell membrane permeable can be measured / determined by methods known in the art and as described herein.

[0365] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0366] The terms "IgG antibody" and "IgG1 antibody" have already been described above. With regard to these definitions and their preferred embodiments, the same as those described above in the context of the ADCs of the invention apply mutatis mutandis.

[0367] The present invention is not limited to a particular antibody, and the ADC can comprise any antibody, preferably any antibody that can be used in cancer therapy.

[0368] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is selected from the group consisting of trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, loncas selected from the group consisting of tuximab, mecbotamab, adecatumumab, D93, gatipotuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab and endrecolomab; and / or The antibody specifically binds to an antigen selected from the group consisting of CD30, Her2 / neu, CD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, NaPi2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, Nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and Nectin-4.

[0369] The above antibodies are known in the art. In the following, the antigen to which each of the antibodies specifically binds is indicated in parentheses: trastuzumab (anti-Her2 / neu), brentuximab (anti-CD30), gemtuzumab (anti-CD33), inotuzumab (anti-CD22), avelumab (anti-PD-L1), cetuximab (anti-EGFR), rituximab (anti-CD20), daratumumab (anti-CD38), pertuzumab (anti-HER2), vedolizumab (anti-integrin alpha 4β7), ocrelizumab (anti-CD20), trastuzumab (anti-IL-6-R), ustekinumab (anti-IL-12 / 23), golimumab (anti-TNFα), obinutuzumab (anti-CD20), sacituzumab (anti-Trop-2), belantamab (anti-BCMA), polatuzumab (anti-CD79b), enfortumab (anti-nectin-4), endrecolomab (anti-EpCAM), gemtuzumab (anti-CD33), loncastuximab (anti-CD 19), mecbotamab (anti-AXL), adecatumumab (anti-EpCAM), D93 (anti-dn-collagen), gatipotuzumab (anti-TA-MUC1), labetuzumab (anti-carcinoembryonic cell adhesion molecule 5), tusamitamab (anti-CEACAM5), upifitamab (anti-NaPi2b), rifastuzumab (anti-NaPi2b), mirvetuximab (anti-FRα), sofituzumab (anti-MUC16), anetumab (anti-mesothelin), tisotumab (anti-TF), cofituzumab (anti-Trop-2), pralzatamab (anti-CD166), radriatuzumab (anti-LIV-1), belantamab (anti-BCMA), patritumab (anti-ERBB3), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), matuzumab (anti-EGFR), portuzumab (anti-HER2), sitatuzumab (anti-TACSTD1), tucotuzumab (anti-EpCAM), and endrecolomab (anti-EpCAM).

[0370] In a particular preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is trastuzumab (anti-Her2 / neu).

[0371] In a preferred embodiment, the antibody is trastuzumab having a heavy chain as set forth in SEQ ID NO:73 and a light chain as set forth in SEQ ID NO:74.

[0372] In another embodiment, the antibody is an anti-Her2 / neu antibody having a heavy chain variable region as set forth in SEQ ID NO:102 and a light chain variable region as set forth in SEQ ID NO:103.

[0373] In another embodiment, the antibody is an anti-Her2 / neu antibody having a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 104, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 105, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 106, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 107, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 108, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 109.

[0374] In another particularly preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is polatuzumab (anti-CD79b).

[0375] In a preferred embodiment, the antibody is polatuzumab having a heavy chain as set forth in SEQ ID NO:71 and a light chain as set forth in SEQ ID NO:72.

[0376] In another embodiment, the antibody is an anti-CD79b antibody having a heavy chain variable region as set forth in SEQ ID NO:110 and a light chain variable region as set forth in SEQ ID NO:111.

[0377] In another embodiment, the antibody is an anti-CD79b antibody having a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 112, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 113, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 114, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 115, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 116, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 117.

[0378] In another particularly preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is enfortumab (anti-nectin-4) or a variant thereof.

[0379] In a preferred embodiment, the antibody is enfortumab having a heavy chain as set forth in SEQ ID NO: 75 and a light chain as set forth in SEQ ID NO: 95, 76 or 77.

[0380] As outlined above, in a preferred embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG antibody.

[0381] Thus, in certain embodiments, the antibody is enfortumab or a variant thereof comprising an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO:94.

[0382] In certain embodiments, the anti-Nectin-4 antibody is enfortumab, which comprises an A at Kabat position 234 and an A at Kabat position 235, with a heavy chain as set forth in SEQ ID NO: 94 and a light chain as set forth in SEQ ID NO: 95, 76, or 77.

[0383] In another embodiment, the antibody is an anti-Nectin-4 antibody having a heavy chain variable region as set forth in SEQ ID NO:118 and a light chain variable region as set forth in SEQ ID NO:119.

[0384] In another embodiment, the antibody is an anti-Nectin-4 antibody having a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 120, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 121, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 122, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 123, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 124 or SEQ ID NO: 142, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 125.

[0385] In another particularly preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is m290 (anti-Nectin-4).

[0386] In a preferred embodiment, the antibody is m290 having a heavy chain as set forth in SEQ ID NO:96 and a light chain as set forth in SEQ ID NO:97.

[0387] In another embodiment, the antibody is an anti-Nectin-4 antibody having a heavy chain variable region as set forth in SEQ ID NO: 126 and a light chain variable region as set forth in SEQ ID NO: 127.

[0388] In another embodiment, the antibody is an anti-Nectin-4 antibody having a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 128, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 129, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 130, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 131, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 132, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 133.

[0389] In another particularly preferred embodiment, the invention relates to an ADC as defined herein, wherein the antibody is upifitamab (anti-NaPi2b).

[0390] In a preferred embodiment, the antibody is upifitamab, having a heavy chain as set forth in SEQ ID NO:98 and a light chain as set forth in SEQ ID NO:99.

[0391] As outlined above, in a preferred embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG antibody.

[0392] Thus, in one particular embodiment, the antibody is upifitamab, which comprises an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO:100.

[0393] In certain embodiments, the anti-NaPi2b antibody is upifitamab, which comprises an A at Kabat position 234 and an A at Kabat position 235, with a heavy chain as set forth in SEQ ID NO: 100 and a light chain as set forth in SEQ ID NO: 101.

[0394] In another embodiment, the antibody is an anti-NaPi2b antibody having a heavy chain variable region as set forth in SEQ ID NO:134 and a light chain variable region as set forth in SEQ ID NO:135.

[0395] In another embodiment, the antibody is an anti-NaPi2b antibody having a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 136, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 137, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 138, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 139, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 140, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 141.

[0396] As mentioned, in certain embodiments, the invention relates to an ADC as defined herein, wherein the antibody is selected from the group consisting of CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collar The antibody specifically binds to an antigen selected from the group consisting of gen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, NaPi2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and nectin-4.

[0397] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4").

[0398] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains two payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0399] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains two payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0400] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of two first payloads and four second payloads (drug-to-antibody ratio of 6, "DAR6").

[0401] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains three payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and two topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0402] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains three payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0403] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of four first payloads and two second payloads (drug-to-antibody ratio of 6, "DAR6").

[0404] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains three payloads: two topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0405] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains three payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0406] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of four first payloads and four second payloads (drug-to-antibody ratio of 8 "DAR8").

[0407] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains four payloads: two topoisomerase I inhibitors that are cell membrane permeable as first payloads; and two topoisomerase I inhibitors that are not cell membrane permeable as second payloads.

[0408] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains four payloads: two camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and two camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0409] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of two first payloads and six second payloads (drug-to-antibody ratio of 8 "DAR8").

[0410] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains four payloads: a single topoisomerase I inhibitor that is cell membrane permeable as the first payload; and three topoisomerase I inhibitors that are not cell membrane permeable as the second payload.

[0411] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains four payloads: a single camptothecin cytotoxic molecule that is cell membrane permeable as the first payload; and three camptothecin cytotoxic molecules that are not cell membrane permeable as the second payload.

[0412] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of six primary payloads and two secondary payloads (drug-to-antibody ratio of 8 "DAR8").

[0413] Thus, in this embodiment, the peptide linker according to the present invention comprises or contains four payloads: three topoisomerase I inhibitors that are cell membrane permeable as the first payload; and a single topoisomerase I inhibitor that is not cell membrane permeable as the second payload.

[0414] Thus, in a preferred embodiment, the peptide linker according to the present invention comprises or contains four payloads: three camptothecin cytotoxic molecules that are cell membrane permeable as the first payload; and a single camptothecin cytotoxic molecule that is not cell membrane permeable as the second payload.

[0415] In a further aspect of the invention, the invention relates to an ADC as defined herein above, wherein the linker further comprises a third payload, preferably a toxin or cytotoxin, more preferably an auristatin, even more preferably MMAE (monomethylauristatin E).

[0416] This further aspect is linked to the ADCs of the invention as defined above and is described in more detail below.

[0417] While the first and second payloads, each as defined herein above, are defined as Topoisomerase I inhibitors in the context of the present invention, in the context of a further embodiment, there is a third payload, which is structurally distinct compared to the first and second payloads.

[0418] Thus, in a preferred embodiment, this third payload is not a topoisomerase I inhibitor that is cell membrane permeable, and is not a topoisomerase I inhibitor that is not cell membrane permeable as defined herein above.

[0419] In a more preferred embodiment, the third payload is not a camptothecin cytotoxic molecule that is cell membrane permeable; and is not a camptothecin cytotoxic molecule that is not cell membrane permeable.

[0420] With respect to the preferred embodiments of the terms "cell membrane permeable topoisomerase I inhibitor," "non-cell membrane permeable topoisomerase I inhibitor," "cell membrane permeable camptothecin cytotoxic molecule," and "non-cell membrane permeable camptothecin cytotoxic molecule," the same applies mutatis mutandis to this further aspect of the invention (except that each feature is not a third payload).

[0421] In a preferred embodiment, the invention according to said further aspect of the invention relates to an ADC as defined herein above, wherein the linker further comprises a third payload, wherein said third payload is a toxin, cytotoxin or cytotoxic molecule.

[0422] The terms "toxin," "cytotoxin," and "cytotoxic molecule" have already been defined above.

[0423] In certain embodiments, in the context of a further aspect, the invention relates to an ADC as defined herein, wherein said linker further comprises a third payload, wherein the third payload is Pyrrolobenzodiazepines (e.g., PBDs); Auristatins (e.g., MMAE, MMAF); Maytansinoids (e.g., maytansine, DM1, DM4, DM21); ·Duocarmycin; · Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; ·Tubulysin; Enediynes (e.g., calicheamicin); Anthracycline derivatives (PNU) (e.g., doxorubicin); · Pyrrole kinesin spindle protein (KSP) inhibitors; · Cryptophycin; Drug efflux pump inhibitors; Sandramycin; antimetabolite or metabolite inhibitors, preferably thymidylate synthase inhibitors; Amanitin (e.g., α-amanitin); DNA damage repair inhibitors; and Topoisomerase II inhibitors At least one selected from the group consisting of:

[0424] That is, in the context of a further aspect, the ADC, preferably the linker of the present invention, preferably comprises a toxin as a third payload. The term "toxin," as used herein, relates to any compound that is toxic to a cell or organism. The terms "poison" or "toxic" are commonly known in the art, and the general term refers to a substance that is harmful or lethal to a living organism in that it kills, damages, or harms the organism or cell and / or induces apoptosis of the cell. Preferably, the toxin is produced by a cell or organism. However, the toxin can also be a chemical derivative or analog of a toxin produced by a cell or organism. The toxin can be, but is not limited to, a small molecule, a peptide, or a protein. Specific examples are neurotoxins, necrotic toxins, hematotoxins, and cytotoxins. In certain embodiments, the toxin is a toxin used in the treatment of neoplastic diseases. That is, the toxin can be conjugated to an antibody in the methods of the present invention and delivered to or into malignant cells due to the targeting specificity of the antibody.

[0425] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin can be an auristatin. As used herein, the term "auristatin" refers to a family of mitotic inhibitors. Auristatins are mitotic inhibitors that are tubulin polymerization blockers. Auristatin derivatives are also included within the definition of the term "auristatin." Thus, in preferred embodiments, the third payload comprises a mitotic inhibitor or a tubulin polymerization blocker.

[0426] Examples of auristatins include, but are not limited to, auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of dolastatins.

[0427] In certain preferred embodiments, the present invention relates to an ADC as defined herein, wherein said ADC, and more particularly said linker, further comprises MMAE (monomethylauristatin E) as a third payload.

[0428] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin can be a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a class of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus and further to maytansinol and the C-3 ester of natural maytansinol (U.S. Pat. No. 4,151,042); synthetic maytansinol C-3 ester analogs (Kupchan et al., J. Med. Chem. 21:31-37, 1978; Higashide et al., Nature 270:721-722, 1977; Kawai ... Kawai et al., J. Med. Chem. 21:31-37, 1978; Higashide et al., Nature 270:721-722, 1977). al., Chem. Farm. Bull. 32:3441-3451; and U.S. Pat. No. 5,416,064); C-3 esters of simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); and C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; and Kawai et al., Chem. Pharm. Bull. 12:3441, 1984). Exemplary maytansinoids that may be used in the methods of the invention or that may be included in the antibody-payload conjugates of the invention are maytansine, DM1, DM3, DM4 and / or DM21.

[0429] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the invention, comprises a toxin as a third payload, and preferably the toxin can be a duocarmycin. Suitable duocarmycins can be, for example, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. The term "duocarmycin" should also be understood to refer to synthetic analogs of duocarmycins, such as adozelesin, bizelesin, carzelesin, KW-2189, and CBI-TMI.

[0430] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin may be a NAMPT inhibitor. As used herein, the terms "NAMPT inhibitor" and "nicotinamide phosphoribosyltransferase inhibitor" refer to inhibitors that reduce the activity of NAMPT. The term "NAMPT inhibitor" may also include prodrugs of NAMPT inhibitors. Examples of NAMPT inhibitors include, but are not limited to, FK866 (also known as APO866), GPP 78 hydrochloride, ST 118804, STF31, pyridyl cyanoguanidine (also known as CH-828), GMX-1778, and P7C3. Additional NAMPT inhibitors are known in the art and may be suitable for use in the compositions and methods described herein. See, e.g., PCT Publication No. WO 2015 / 054060, U.S. Patent Nos. 8,211,912, and 9,676,721, which are incorporated by reference in their entireties. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

[0431] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, preferably a tubulysin. Tubulysin is a cytotoxic peptide containing nine members (A-I). Tubulysin A has potential use as an anticancer drug. It arrests cells in the G2 / M phase. Tubulysin A inhibits polymerization more efficiently than vinblastine and induces depolymerization of isolated microtubules. Tubulysin A has potent cytostatic effects against various tumor cell lines with an IC50 in the picomolar concentration range. Another tubulysin that can be used in the methods of the present invention can be tubulysin E.

[0432] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the invention, comprises a toxin as a third payload, and preferably the toxin can be an enediyne. The term "enediyne," as used herein, refers to a class of bacterial natural products characterized by either a 9- or 10-membered ring containing two triple bonds separated by a double bond (see, e.g., KC Nicolaou; A.L. Smith; E.W. Yue (1993). "Chemistry and biology of natural and designed enediynes." PNAS 90(13):5881-5888; the entire contents of which are incorporated herein by reference). Some enediynes can undergo Bergmann cyclization, and the resulting diradical, a 1,4-dehydrobenzene derivative, can remove a hydrogen atom from the sugar backbone of DNA, resulting in DNA strand cleavage (see, e.g., S. Walker; R. Landovitz; W.D. Ding; G.A. Ellestad; D. Kahne (1992). "Cleavage behavior of calicheamicin gamma 1 and calicheamicin T." Proc. Natl. Acad. Sci. USA 89(10):4608-12; the entire contents of which are incorporated herein by reference). Their reactivity with DNA confers antibiotic properties to many enediynes, and several enediynes are under clinical investigation as anticancer antibiotics. Non-limiting examples of enediynes include dynemicin, neocarzinostatin, calicheamicin, and esperamicin (see, e.g., Adrian L. Smith and KC Bicolaou, "The Enediyne Antibiotics," J. Med. Chem., 1996, 39(11), pp. 2103-2117; and Donald Borders, "Enediyne antibiotics as antitumor agents," Informa Healthcare; 1st edition (Nov. 23, 1994, ISBN-10:0824789385; the entire contents of which are incorporated herein by reference).In certain embodiments, the toxin can be calicheamicin.

[0433] In certain embodiments, in the context of further aspects, the ADC, preferably a linker of the invention, comprises a toxin as a third payload, and preferably the toxin can be doxorubicin. "Doxorubicin," as used herein, refers to a member of the anthracycline family derived from the Streptomyces bacterium Streptomyces peucetius var. caesius, and includes doxorubicin, daunorubicin, epirubicin, and idarubicin.

[0434] In certain embodiments, in the context of a further aspect, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin can be a kinesin spindle protein inhibitor. The term "kinesin spindle protein inhibitor" refers to a compound that inhibits kinesin spindle protein, which is involved in the assembly of bipolar spindles during cell division. Kinesin spindle protein inhibitors are being investigated for the treatment of cancer. Examples of kinesin spindle protein inhibitors include ispinesib. Further, the term "kinesin spindle protein inhibitor" includes SB715992 or SB743921 from GlaxoSmithKline and pentamidine / chlorpromarin from CombinatoRx.

[0435] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the invention, comprises a toxin as a third payload, preferably the toxin may be cryptophycin or a derivative as described in U.S. Patent Application Publication No. 20180078656A1, U.S. Patent Application Publication No. 20210163458A1, U.S. Patent Application Publication No. 20210228726A1, which are incorporated by reference.

[0436] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the invention, comprises a toxin as a third payload, which may preferably be sandramycin, a depsipeptide originally isolated from Nocardioides sp. (ATCC 39419) that has been shown to have cytotoxic and antitumor activity.

[0437] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin can be an antimetabolite or a metabolite inhibitor. Antimetabolites and metabolite inhibitors are known in the art. As an example, the toxin can be pemetrexed. Pemetrexed is a chemotherapeutic agent in the class of chemotherapy drugs called antifolates. It is known to inhibit three enzymes used in purine and pyrimidine synthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal and cancer cells.

[0438] In certain other embodiments, any other antimetabolite / metabolite inhibitor may be used that inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), and / or glycinamide ribonucleotide formyltransferase (GARFT).

[0439] In certain other embodiments, the antimetabolite / metabolite inhibitor is a thymidine synthase (or thymidylate synthase) inhibitor. Thymidylate synthase inhibitors are chemical agents that inhibit the enzyme thymidylate synthase and have potential as anti-cancer chemotherapy. This inhibition prevents methylation of C5 of deoxyuridine monophosphate (dUMP), thereby inhibiting the synthesis of deoxyuridine monophosphate (dTMP). A downstream effect is the promotion of cell death, as cells cannot properly undergo DNA synthesis if they lack dTMP, a necessary precursor of dTTP. Within the present invention, thymidylate synthase inhibitors can be, but are not limited to, raltitrexed, pemetrexed, nolatrexed, ZD9331, GS7904L, fluorouracil, BGC-945, and OSI-7904L.

[0440] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, and preferably the toxin can be an amatoxin. Amatoxins (including alpha-amanitin, beta-amanitin, and amanitin) are cyclic peptides composed of eight amino acids. They can be isolated from the Amanita phalloides mushroom or prepared synthetically from the components. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby inhibiting transcription and protein biosynthesis in affected cells. Inhibition of transcription in cells causes growth and proliferation to cease. Although not covalently bound, the complex between amanitin and RNA polymerase II is very strong (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, making recovery of affected cells unlikely. If transcription is inhibited in cells for too long, the cells will undergo programmed cell death (apoptosis). In a preferred embodiment, the term "amatoxin" as used herein refers to alpha-amatoxin or variants thereof as described, for example, in WO 2010 / 115630, WO 2010 / 115629, WO 2012 / 119787, WO 2012 / 041504, and WO 2014 / 135282.

[0441] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the present invention, comprises a DNA damage repair inhibitor as a third payload. DNA repair inhibitors are known in the art. Any DNA repair inhibitor known in the art can be used as a third payload. Without being bound by theory, some examples of DNA repair inhibitors are PARP inhibitors, ATR / ATM inhibitors and Chk1 inhibitors. Furthermore, as a DNA repair inhibitor, a kinase inhibitor can be used as a third payload. In a preferred embodiment, the kinase inhibitor is a tyrosine kinase inhibitor.

[0442] In certain embodiments, in the context of a further aspect, the ADC, preferably the linker of the present invention, comprises a toxin as a third payload, preferably a topoisomerase II inhibitor. Topoisomerase II inhibitors are known in the art. In a preferred embodiment, the topoisomerase II inhibitor that can be used as the third payload can be selected from the group consisting of aminocoumarin (novobiocin), fluoroquinolone (cinonaxin), epipodophyllotoxin derivatives (etoposide), acridine or anthraquinone derivatives (amsacrine, pixantrone).

[0443] In certain embodiments, in the context of further aspects, the ADC, preferably the linker of the invention, consists of or comprises one or more third payloads.

[0444] In embodiments in which an ADC, preferably a linker of the invention, consists of or comprises two or more tertiary payloads, the two or more tertiary payloads are identical in structure. In preferred embodiments, the two or more tertiary payloads that are identical in structure are as defined herein above.

[0445] In another preferred embodiment, the two or more third payloads differ in structure. In a preferred embodiment, the two or more structurally different payloads are one or more of the third payloads as defined herein above.

[0446] While the number of tertiary payloads is not limited, in preferred embodiments, in the context of further aspects, the linker of the present invention consists of or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tertiary payloads. In certain preferred embodiments, the linker of the present invention consists of one tertiary payload.

[0447] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the third payload is linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue comprised in the peptide linker; preferably The third payload is linked to the C-terminus of the peptide linker.

[0448] In a preferred embodiment, the present invention relates to an ADC as defined herein, wherein the first payload and / or the second payload and / or the third payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue comprised in the peptide linker; preferably the first payload, the second payload, and the third payload are linked to the N-terminus of a peptide linker; or the first payload, the second payload, and the third payload are linked to the C-terminus of the peptide linker; the first payload and the second payload are linked to the N-terminus of the peptide linker and the third payload is linked to the C-terminus of the peptide linker; or the first payload and the third payload are linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker; or the second payload and the third payload are linked to the N-terminus of the peptide linker and the first payload is linked to the C-terminus of the peptide linker; or the first payload is linked to the N-terminus of the peptide linker and the second payload and the third payload are linked to the C-terminus of the peptide linker; or the second payload is linked to the N-terminus of the peptide linker and the first payload and the third payload are linked to the C-terminus of the peptide linker; or The third payload is linked to the N-terminus of the peptide linker and the first payload and second payload are linked to the C-terminus of the peptide linker.

[0449] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker has the following structure: [Payload 1]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[payload 2] / [payload 3]; or [Payload 1] / [Payload 3]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 2]; or [Payload 2]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[payload 1] / [payload 3]; or [Payload 2] / [Payload 3]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 1]; or [Payload 1] / [Payload 2]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 3]; or [Payload 3]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 1] / [Payload 2] (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; [Payload 3] is the third payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z is absent or is a spacer comprising an alkyl or heteroalkyl group, preferably the spacer is (CH2)2; and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0450] In certain embodiments, the invention relates to an ADC according to the invention, wherein two payloads are linked to the same functional group of the peptide linker, preferably via a chemical linker comprising a disubstituted amine.

[0451] That is, in a preferred embodiment, the invention relates to an ADC according to the invention, wherein the linker has the structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -Z3-X-[payload]; or [Payload]-X-Z1-(Aa) m -Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; or ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[Payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, n, o, p and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and even more preferably, n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxylic acid linker, and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0452] In an even more preferred embodiment, the invention relates to an ADC according to the invention, wherein the linker has the structure: ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[payload]; or [Payload]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2; or ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, p, and p* are integers ranging from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m Or the N-terminus of the disubstituted amine (N) is (Aa) n or (Lys); and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0453] The linker preferably comprises at least one additional positively charged amino acid residue in addition to the lysine residue, as described in detail elsewhere herein. That is, in a particular aspect, the invention relates to an ADC according to the invention, comprising (Aa) n -(Lys)-(Aa) o comprises the sequence motif Arg-Lys(RK) or His-Lys(HK) (N->C orientation). In another particular embodiment, the invention relates to an ADC according to the invention, comprising (Aa) n -(Lys)-(Aa) o is or includes RK or RKAA (N->C direction).

[0454] In a preferred embodiment, the present invention relates to an ADC as defined herein, wherein said ADC comprises two or more first payloads and / or two or more second payloads and / or two or more third payloads.

[0455] As already outlined above, the peptide linker according to the present invention comprises or contains at least two payloads, namely a "first payload" and a "second payload".

[0456] In embodiments in which a "third payload" is included, a peptide linker according to the invention comprises or contains at least three payloads.

[0457] When the peptide linker comprises or contains only three payloads, these three payloads are structurally different, i.e., the first payload is a camptothecin cytotoxic molecule that is cell membrane permeable; and the second payload is a camptothecin cytotoxic molecule that is not cell membrane permeable; and the third payload is not a camptothecin derivative cytotoxic molecule but is preferably a toxin, preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, more preferably MMAE as defined herein.

[0458] When a peptide linker comprises or contains four or more payloads, the payloads may be identical or different in structure, while at least two of the payloads may be identical in structure.

[0459] By coupling two or more identical payloads to a peptide linker, it becomes possible to increase the concentration of the payload in the target tissue or cell of the antibody-payload conjugate. For example, when the peptide linker of the antibody-payload conjugate contains two or more identical toxins (resulting in a DAR>4 ADC), the concentration of the toxin in the target tissue or cell is increased compared to a conventional DAR2 ADC. Using the peptide linkers of the present invention, ADCs containing 4, 6, or 8 identical payload molecules may be obtained.

[0460] In embodiments where a third payload is included, in certain embodiments, a peptide linker according to the invention comprises 3 to 6 payloads.

[0461] In certain embodiments, the peptide linker according to the present invention comprises or contains three payloads: as a first payload, a single topoisomerase I inhibitor that is cell membrane permeable; as a second payload, a single topoisomerase I inhibitor that is not cell membrane permeable; and as a third payload, a single toxin, preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, more preferably MMAE as defined herein.

[0462] Further embodiments corresponding to the above embodiments are preferred, in which the peptide linker according to the invention comprises 2 to 4 payloads, but is supplemented with 1 or 2 or 3 third payloads.

[0463] In embodiments in which a third payload is included, these three payloads defined above can be coupled to the peptide linker in different ways.

[0464] As mentioned above, the inventors have demonstrated different methods for coupling two or more payloads to a peptide linker.

[0465] In certain embodiments in which a third payload is included, the first payload and / or the second payload and / or the third payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue included in the peptide linker.

[0466] In certain embodiments where a third payload is included, two or three payloads may be coupled to the C-terminus of the peptide linker. In other embodiments, two or three payloads may be coupled to the N-terminus of the peptide linker. In yet other embodiments, one, two, or three payloads may be coupled to the N-terminus of the peptide linker and the C-terminus of the peptide linker, respectively.

[0467] In certain embodiments where a third payload is included, three payloads may be coupled to the C-terminus of the peptide linker. In other embodiments, three payloads may be coupled to the N-terminus of the peptide linker. In yet other embodiments, one, two, or three payloads may be coupled to the N-terminus of the peptide linker and the C-terminus of the peptide linker, respectively.

[0468] In another specific embodiment in which a third payload is included, one or more payloads are attached to the N-terminus of the amine-containing peptide linker and one or more payloads are attached to the C-terminus of the amine-containing peptide linker.

[0469] In another specific embodiment where a third payload is included, two payloads are attached to the N-terminus of the peptide linker and one payload is attached to the C-terminus of the peptide linker. An example of a corresponding configuration is shown in Figure 8.

[0470] In certain embodiments, one, two, or three payloads may each be attached to the N-terminus of an amine-containing peptide linker, and one, two, or three payloads may each be attached to the C-terminus of said amine-containing peptide linker.

[0471] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the third payload is MMAE.

[0472] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein the linker has the following structure: [ka] It comprises or consists of:

[0473] In a preferred embodiment, the invention relates to an ADC as defined herein, wherein said ADC consists of two first payloads, two second payloads, and two tertiary payloads (drug-to-antibody ratio of 6, "DAR6").

[0474] Thus, in this embodiment, the peptide linker according to the invention can carry three payloads, namely: As the first payload: a cell membrane-permeable topoisomerase I inhibitor; and As a second payload: one topoisomerase I inhibitor that is not cell membrane permeable; and One tertiary payload: Preferably, as the third payload, one toxin, preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, more preferably MMAE as defined herein. It comprises or contains.

[0475] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"); and The antibody relates to an ADC as defined herein having an IgG, preferably an IgG1 antibody.

[0476] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0477] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and As a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; and a third payload as defined above, preferably a toxin, more preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, even more preferably MMAE; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads, two second payloads, and two tertiary payloads (a drug-to-antibody ratio of 6, "DAR6"); and The antibody relates to an ADC as defined herein having an IgG, preferably an IgG1 antibody.

[0478] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0479] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"); and The antibody relates to an ADC as defined herein having a cytotoxicity associated with trastuzumab.

[0480] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0481] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"); and The antibody relates to an ADC as defined herein having an antibody that specifically binds to the antigen NaPi2b.

[0482] In the context of this particular preferred embodiment, in an even more preferred embodiment, the linker has the following structure: [ka] It comprises or consists of:

[0483] In certain preferred embodiments, the present invention relates to an ADC comprising an antibody that specifically binds to the antigen NaPi2b, Preferably, the antibody comprises a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 136, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 137, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 138, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 139, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 140, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 141; More preferably, the antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 134 and a light chain variable region as set forth in SEQ ID NO: 135; Even more preferably, the antibody comprises a heavy chain as set forth in SEQ ID NO: 98 and a light chain as set forth in SEQ ID NO: 99 or a heavy chain as set forth in SEQ ID NO: 100 and a light chain as set forth in SEQ ID NO: 101; and the linker has the structure: [ka] and The linker is conjugated via a lysine residue to glutamine residue Q295 (EU numbering) of the antibody heavy chain.

[0484] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0485] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"); and The antibody relates to an ADC as defined herein having a nucleotide sequence that specifically binds to the antigen Nectin-4.

[0486] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0487] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and As a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; and a third payload as defined above, preferably a toxin, more preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, even more preferably MMAE; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads, two second payloads, and two tertiary payloads (a drug-to-antibody ratio of 6, "DAR6"); and The antibody relates to an ADC as defined herein having a cytotoxicity associated with trastuzumab.

[0488] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0489] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and As a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; and a third payload as defined above, preferably a toxin, more preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, even more preferably MMAE; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads, two second payloads, and two tertiary payloads (a drug-to-antibody ratio of 6, "DAR6"); and The antibody relates to an ADC as defined herein having an antibody that specifically binds to the antigen NaPi2b.

[0490] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0491] In certain preferred embodiments, the present invention provides a compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and As a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; and a third payload as defined above, preferably a toxin, more preferably a mitotic inhibitor, a tubulin polymerization blocker, and / or an auristatin, even more preferably MMAE; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads, two second payloads, and two tertiary payloads (a drug-to-antibody ratio of 6, "DAR6"); and The antibody relates to an ADC as defined herein having an antibody that specifically binds to the antigen Nectin-4, preferably the antibody is m290 as defined herein.

[0492] In the context of this particular preferred embodiment, in an even more preferred embodiment, the linker has the following structure: [ka] It comprises or consists of:

[0493] In certain preferred embodiments, the present invention relates to an ADC comprising an antibody that specifically binds to the antigen Nectin-4, Preferably, the antibody comprises a heavy chain CDR1 (CDR-H1, Kabat) as set forth in SEQ ID NO: 128, a heavy chain CDR2 (CDR-H2, Kabat) as set forth in SEQ ID NO: 129, a heavy chain CDR3 (CDR-H3, Kabat) as set forth in SEQ ID NO: 130, a light chain CDR1 (CDR-L1, Kabat) as set forth in SEQ ID NO: 131, a light chain CDR2 (CDR-L2, Kabat) as set forth in SEQ ID NO: 132, and a light chain CDR3 (CDR-L3, Kabat) as set forth in SEQ ID NO: 133; More preferably, the antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 126 and a light chain variable region as set forth in SEQ ID NO: 127; Even more preferably, the antibody comprises a heavy chain as set forth in SEQ ID NO: 96 and a light chain as set forth in SEQ ID NO: 97; and the linker has the structure: [ka] and The linker is conjugated via a lysine residue to glutamine residue Q295 (EU numbering) of the antibody heavy chain.

[0494] With regard to the definition of this ADC and its preferred embodiments, the same as stated above in the context of the ADC of the present invention applies mutatis mutandis.

[0495] The ADCs of the present invention are particularly useful in medical settings.

[0496] In a preferred embodiment, the invention relates to a pharmaceutical composition comprising an ADC as defined herein and at least one pharmaceutically acceptable ingredient.

[0497] The term "pharmaceutical composition," as used herein, refers to any composition containing chemicals or active ingredients, wherein the composition is intended for use in the medical cure, treatment, or prevention of disease and is in a form that allows the active ingredient to be effective. In particular, a pharmaceutical composition does not contain excipients that are unacceptably toxic to the subject to whom the composition will be administered. A pharmaceutical composition is sterile, i.e., aseptic and free of all living microorganisms and their spores. The pharmaceutical composition of the present invention is preferably liquid.

[0498] A pharmaceutical composition according to the present invention comprises an antibody-drug conjugate as disclosed herein. The pharmaceutical composition comprising the antibody-drug conjugate is preferably used for the treatment of a disease.

[0499] A pharmaceutical composition according to the present invention may comprise at least one pharmaceutically acceptable ingredient.

[0500] Pharmaceutically acceptable ingredients refer to ingredients in a pharmaceutical formulation, other than the active ingredient, that are non-toxic to a subject. Pharmaceutically acceptable ingredients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0501] Pharmaceutical formulations of the antibody-payload conjugates described herein are prepared by mixing such conjugates having the desired degree of purity with one or more optional pharmaceutically acceptable components (Flemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable components are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable ingredients herein further include intercalating drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. For example, a sHASEGP can be combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0502] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the present invention comprising at least one additional therapeutically active agent.

[0503] The pharmaceutical composition comprising antibody-payload conjugate can comprise one or more therapeutically effective drugs.It should be understood that antibody-payload conjugate can be used in various therapeutic agents.Therefore, the additional therapeutically effective drug in the pharmaceutical composition can vary depending on the use of the pharmaceutical composition.

[0504] In certain embodiments, as outlined in further more detail below, pharmaceutical compositions comprising an antibody-payload conjugate according to the invention may be used in the treatment of neoplastic disorders, preferably in the treatment of cancer. In such embodiments, the pharmaceutical composition may comprise one or more additional anti-cancer agents. The term "anti-cancer" agent is used herein to refer to one or a combination of drugs conventionally used to treat cancer.

[0505] For example, a pharmaceutical composition comprising an antibody-payload conjugate according to the invention may further comprise one or more chemotherapeutic agents. As used herein, the term "chemotherapeutic agent" or "chemotherapeutic drug" or "chemotherapeutic agent" refers to an agent that can be used in a pharmaceutically effective amount to reduce, prevent, reduce, limit, and / or slow the growth of metastases or neoplasms, or to directly kill neoplastic cells by neoplastic necrosis or apoptosis or any other mechanism, or otherwise reduce, prevent, reduce, limit, and / or slow the growth of metastases or neoplasms in a subject with a neoplastic disease. Chemotherapeutic agents include, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; antifolates, platinum agents; anthracyclines / anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormone conjugates; antihormones; enzymes, proteins, peptides and polyclonal and / or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; microtubule inhibitors; alkylating agents; antimetabolites; topoisomerase inhibitors; antivirals; and various other cytotoxic and cytostatic agents.

[0506] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in therapy.

[0507] Therefore, the antibody-payload conjugate or pharmaceutical composition according to the present invention can be used in the treatment of a subject / patient. The individual, subject, or patient is preferably a mammal. Mammals include, but are not limited to, livestock (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0508] In a preferred embodiment, the invention relates to an ADC as defined herein for use in a method of treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic disease.

[0509] Neoplastic disease or neoplasm, in its broadest sense, is generally understood as a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. This abnormal growth usually forms a mass, which may be called a tumor or tumour. ICD-10 classifies neoplasms into four major groups: benign neoplasms, neoplasms in situ, malignant neoplasms, and neoplasms of a specific or unknown behaviour. Malignant neoplasms are also known simply as cancer. Neoplasms can be benign, potentially malignant, or malignant (cancer).

[0510] A patient suffering from cancer may be a patient who has not been previously treated with any anti-cancer therapy, however, a patient suffering from cancer may also be a patient who was refractory to a previous anti-cancer treatment.

[0511] The term "neoplastic disease" as used herein refers to a condition characterized by uncontrolled, abnormal cell growth. Neoplastic diseases include cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatocellular carcinoma, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, and peripheral neuroepithelioma. Preferred cancers include liver cancer, lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, and peripheral neuroepithelioma.

[0512] The antibody-payload conjugate according to the present invention is preferably used for the treatment of cancer. Therefore, in certain embodiments, the antibody-payload conjugate according to the present invention comprises an antibody that specifically binds to an antigen present on a tumor cell. In certain embodiments, the antigen may be an antigen on the surface of a tumor cell. In certain embodiments, the antigen on the surface of a tumor cell may be internalized into the cell together with the antibody-payload conjugate upon binding of the antibody-payload conjugate to the antigen.

[0513] In a preferred embodiment, the invention relates to an ADC as defined herein or a pharmaceutical composition as defined herein, the antibody-payload conjugate comprises trastuzumab, and the neoplastic disease is HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer; the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell related cancer; preferably, the B-cell related cancer is non-Hodgkin's lymphoma, in particular, the B-cell related cancer is diffuse large B-cell lymphoma; or the antibody-payload conjugate comprises enfortumab or an enfortumab variant, or m290, and the neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer; or The antibody-payload conjugate comprises upifitamab, and the neoplastic disease is NaPi2b-positive cancer, particularly NaPi2b-positive lung cancer or ovarian cancer.

[0514] Thus, in certain embodiments, the antibody-payload conjugates and / or pharmaceutical compositions according to the invention may be used in the treatment of HER2-positive cancers.

[0515] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the present invention, wherein the antibody-payload conjugate comprises trastuzumab and the neoplastic disease is a HER2-positive cancer, in particular a HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

[0516] To this end, the antibody-payload conjugate comprises an anti-HER2 / neu antibody as disclosed herein, preferably one that is internalized into the target cell upon binding to HER2 / neu. In certain embodiments, the anti-HER2 / neu antibody is trastuzumab, having a heavy chain as set forth in SEQ ID NO:73 and a light chain as set forth in SEQ ID NO:74.

[0517] In certain embodiments, the anti-HER2 / neu antibody comprised in the antibody-payload conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in Figures 4, 6, 7, or 8 or any one of the linkers disclosed herein.

[0518] As used herein, HER2-positive cancer may be, but is not limited to, HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer. Those skilled in the art can determine whether a cancer is HER2-positive cancer. For example, tumor cells may be isolated in a biopsy, and the presence of HER2 / neu may be determined by any method known in the art.

[0519] Additionally, the anti-HER2 / neu antibody-payload conjugate and / or pharmaceutical compositions comprising the anti-HER2 / neu antibody-payload conjugate may be used in combination with other therapies suitable for the treatment of HER2-positive cancers.

[0520] Thus, in a particular embodiment, the invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-payload conjugate or pharmaceutical composition is administered in combination with lapatinib, capecitabine and / or a taxane.

[0521] It should be understood that the antibody-payload conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with the additional therapeutic agent, such as lapatinib, capecitabine, and / or a taxane. Instead, the antibody-payload conjugate or pharmaceutical composition may be administered on a different dosing schedule, and consequently on different days, than other therapeutic agents used to treat the same disease.

[0522] In certain embodiments, the antibody-payload conjugates and / or pharmaceutical compositions according to the invention may be used in the treatment of B-cell related cancers.

[0523] Thus, in a particular embodiment, the invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell related cancer.

[0524] To this end, the antibody-payload conjugate comprises an anti-CD79b antibody as disclosed herein, preferably one that is internalized into target cells upon binding to CD79b. In certain embodiments, the anti-CD79b antibody is polatuzumab, having a heavy chain as set forth in SEQ ID NO:71 and a light chain as set forth in SEQ ID NO:72.

[0525] In certain embodiments, the anti-CD79b antibody comprised in the antibody-payload conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in Figures 4, 6, 7, or 8 or any one of the linkers disclosed herein.

[0526] B-cell related cancers include high-grade, intermediate-grade, and low-grade lymphomas (e.g., B-cell lymphomas such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma and T-cell lymphoma) and leukemias (secondary leukemia, B-cell leukemia (CD5+ B lymphocytes)), etc. the cancer may be any one selected from the group consisting of other blood and / or B-cell or T-cell cancers, including cancers of polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes and natural killer cells, myeloid leukemias such as chronic lymphocytic leukemia (CLL), acute myeloid leukemia, lymphoid leukemias such as chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) and myelodysplasia, and cancers of further hematopoietic cells including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes and natural killer cells. Also included are cancerous B-cell proliferative disorders selected from the following: lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0527] In a particular embodiment, the invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the invention, wherein the B-cell related cancer is non-Hodgkin's lymphoma, in particular the B-cell related cancer is diffuse large B-cell lymphoma.

[0528] Additionally, the anti-CD79b antibody-payload conjugates and / or pharmaceutical compositions comprising anti-CD79b antibody-payload conjugates may be used in combination with other therapies suitable for the treatment of B-cell related cancers.

[0529] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the present invention, wherein the antibody-payload conjugate or pharmaceutical composition is administered in combination with bendamustine and / or rituximab.

[0530] It should be understood that the antibody-payload conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with the additional therapeutic agent, such as bendamustine and / or rituximab. Instead, the antibody-payload conjugate or pharmaceutical composition may be administered on a different dosing schedule, and consequently on a different day, than other therapeutic agents used to treat the same disease.

[0531] In certain embodiments, the antibody-payload conjugates and / or pharmaceutical compositions according to the present invention may be used in the treatment of Nectin-4 positive cancers.

[0532] That is, in a particular embodiment, the present invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the present invention, wherein the antibody-payload conjugate comprises enfortumab or an enfortumab variant, or m290, and the neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

[0533] To this end, the antibody-payload conjugate comprises an anti-Nectin-4 antibody as disclosed herein, preferably one that is internalized into target cells upon binding to Nectin-4. In certain embodiments, the anti-Nectin-4 antibody is enfortumab, having a heavy chain as set forth in SEQ ID NO: 75 and a light chain as set forth in SEQ ID NO: 95, 76, or 77.

[0534] Furthermore, as outlined above, in a preferred embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG antibody.

[0535] Thus, in certain embodiments, the antibody is enfortumab or a variant thereof comprising an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO:94.

[0536] In certain embodiments, the antibody is enfortumab comprising an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO: 94 and a light chain as set forth in SEQ ID NO: 95.

[0537] In certain other embodiments, the invention relates to an ADC as defined herein, wherein the antibody is m290 (anti-Nectin-4).

[0538] In a preferred embodiment, the antibody is m290 having a heavy chain as set forth in SEQ ID NO:96 and a light chain as set forth in SEQ ID NO:97.

[0539] In certain embodiments, the anti-Nectin-4 antibody included in the antibody-payload conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in Figures 4, 6, 7, or 8 or any one of the linkers disclosed herein. In a preferred embodiment, the anti-Nectin-4 antibody, particularly the anti-Nectin-4 antibody m290, may be conjugated to the linker shown in Figure 8.

[0540] As used herein, a Nectin-4-positive cancer may be, but is not limited to, a Nectin-4-positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer. Those skilled in the art can determine whether a cancer is a Nectin-4-positive cancer. For example, tumor cells may be isolated in a biopsy, and the presence of Nectin-4 may be determined by any method known in the art.

[0541] The anti-Nectin-4 antibody-payload conjugate and / or pharmaceutical composition comprising the anti-Nectin-4 antibody-payload conjugate according to the present invention may be administered alone in patients who have previously received a PD-1 or PD-L1 inhibitor in combination with a platinum-based chemotherapy agent before or after surgery.

[0542] Furthermore, the anti-Nectin-4 antibody-payload conjugate and / or pharmaceutical composition comprising the anti-Nectin-4 antibody-payload conjugate may be used in combination with other therapies suitable for the treatment of Nectin-4-positive cancers.

[0543] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the present invention, wherein the antibody-payload conjugate or the pharmaceutical composition is administered in combination with a platinum-based chemotherapeutic agent and / or pembrolizumab.

[0544] It should be understood that the antibody-payload conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with the additional therapeutic agent, such as a cisplatin-based chemotherapeutic agent and / or pembrolizumab. Instead, the antibody-payload conjugate or pharmaceutical composition may be administered on a different schedule, and consequently on a different day, than other therapeutic agents used to treat the same disease.

[0545] In certain embodiments, the antibody-payload conjugates and / or pharmaceutical compositions according to the invention may be used in the treatment of NaPi2b-positive cancers.

[0546] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the present invention, wherein the antibody-payload conjugate comprises upifitamab and the neoplastic disease is an anti-NaPi2b-positive cancer.

[0547] To this end, the antibody-payload conjugate comprises an anti-NaPi2b antibody as disclosed herein, preferably one that is internalized into target cells upon binding to NaPi2b. In a preferred embodiment, the antibody is upifitamab, having a heavy chain as set forth in SEQ ID NO:98 and a light chain as set forth in SEQ ID NO:99.

[0548] As outlined above, in a preferred embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the antibody comprises an A at Kabat position 234 and / or an A at Kabat position 235, preferably an IgG antibody.

[0549] Thus, in one particular embodiment, the antibody is upifitamab, which comprises an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO:100.

[0550] In certain embodiments, the antibody is upifitamab comprising an A at Kabat position 234 and an A at Kabat position 235 with a heavy chain as set forth in SEQ ID NO: 100 and a light chain as set forth in SEQ ID NO: 101.

[0551] In certain embodiments, the anti-NaPi2b antibody included in the antibody-payload conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in Figures 4, 6, or 7, or any one of the linkers disclosed herein. In preferred embodiments, the anti-NaPi2b antibody, particularly the anti-NaPi2b antibody upifitamab, may be conjugated to any one of the linkers shown in Figure 4.

[0552] As used herein, NaPi2b-positive cancer may be, but is not limited to, NaPi2b-positive lung cancer or ovarian cancer.Those skilled in the art can determine whether a cancer is a NaPi2b-positive cancer.For example, tumor cells may be isolated in a biopsy, and the presence of NaPi2b may be determined by any method known in the art.

[0553] In a particular embodiment, the present invention relates to the use of an antibody-payload conjugate according to the invention, or a pharmaceutical composition according to the invention, for the manufacture of a medicament for the treatment of a patient suffering from, at risk of developing and / or diagnosed with a neoplastic disease.

[0554] With regard to preferred embodiments of the use of an antibody-payload conjugate according to the invention, or a pharmaceutical composition according to the invention, for the manufacture of a medicament for the treatment of a patient suffering from, at risk of developing and / or diagnosed with a neoplastic disease, the same applies mutatis mutandis as stated above in the context of the composition for use or the pharmaceutical composition as defined above.

[0555] The antibody-payload conjugates or pharmaceutical compositions according to the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional, intrauterine, or intravesical administration, if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0556] The antibody-payload conjugates or pharmaceutical compositions of the present invention may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to physicians. The antibody-payload conjugates or pharmaceutical compositions of the present invention need not, but are optionally, formulated with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs depends on the amount of antibody-payload conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and via any route of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0557] The appropriate dosage of an antibody-payload conjugate or pharmaceutical composition according to the invention (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the type of antibody-payload conjugate, the severity and course of the disease, whether the antibody-linker conjugate is administered prophylactically or therapeutically, previous therapy, the patient's medical history and response to the antibody-linker conjugate, and the discretion of the attending physician. The antibody-payload conjugate or pharmaceutical composition according to the invention is suitably administered to the patient at one time or over a series of treatments.

[0558] The present invention also relates to a method of treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic disease, wherein an ADC or pharmaceutical composition of the invention as defined herein is administered to the subject, preferably in a therapeutically effective amount as defined herein.

[0559] With regard to the preferred embodiment of the method for treatment, the same applies mutatis mutandis as stated above in the context of the composition for use or the pharmaceutical composition as defined above.

[0560] Another aspect of the present invention relates to such linkers, particularly the peptide linkers disclosed herein.

[0561] That is, in certain embodiments, the present invention relates to a linker comprising a first and a second payload, wherein the first payload is a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and the second payload is a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable; Preferably, the linker is for conjugation to an antibody.

[0562] In another embodiment, the invention relates to a linker according to the present invention, wherein the camptothecin is exatecan or deruxtecan.

[0563] In another embodiment, the present invention relates to a linker according to the present invention, wherein the second payload is modified to reduce its cell membrane permeability.

[0564] In another embodiment, the present invention relates to a linker according to the present invention, wherein a second payload comprises a glycine residue linked to said topoisomerase I inhibitor of the second payload.

[0565] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker is a peptide linker.

[0566] In another embodiment, the present invention relates to a linker according to the present invention, wherein the first payload and / or the second payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue comprised in the peptide linker; preferably The first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa.

[0567] In another embodiment, the invention relates to a linker according to the invention, wherein the linker comprises a primary amine for conjugation to an antibody, preferably the primary amine is comprised in a lysine residue, a lysine mimetic or a lysine derivative; or the primary amine has the structure NH2-(CH2) 1~10 It is comprised in an amino acid residue having a -COOH group; preferably, the amino acid residue is comprised in a peptide linker.

[0568] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker further comprises at least one positively charged amino acid residue, preferably wherein the at least one positively charged amino acid residue is selected from arginine and / or histidine.

[0569] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the following structure: [Payload 1]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 2]; or [Payload 2]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2; and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0570] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) m +(Aa) n +(Aa) o is >0, preferably (Aa) n +(Aa) o is >0.

[0571] In another embodiment, the present invention relates to a linker according to the present invention, wherein Z2 is (Aa) m The N-terminus of (Aa) n or (Lys) is a dicarboxylic acid linked to the N-terminus of (Aa); and one payload is directly or indirectly linked to (Aa) mThe other payload is directly or indirectly linked to the C-terminus of (Lys) or (Aa) o or vice versa.

[0572] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the following structure: [Payload 1]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[Payload 2]; or [Payload 2]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa)oX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer ranging from 1 to 10, preferably from 1 to 6, more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m The N-terminus of (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0573] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) n -(Lys)-(Aa) o contains the sequence motif Arg-Lys (RK) or His-Lys (HK) (N->C orientation).

[0574] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) n -(Lys)-(Aa) o is or includes RK or RKAA (N->C direction).

[0575] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the following structure: [ka] It comprises or consists of:

[0576] In another embodiment, the present invention relates to a linker according to the present invention, wherein said linker further comprises a third payload, preferably a toxin or cytotoxin, more preferably an auristatin, even more preferably MMAE (monomethylauristatin E).

[0577] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker is a peptide linker and the third payload is linked to the N- or C-terminus of the peptide linker, or the third payload is linked to a side chain of an amino acid residue contained in the peptide linker.

[0578] In another embodiment, the present invention relates to a linker according to the present invention, wherein two payloads are linked to the same functional group of the peptide linker, preferably via a chemical linker comprising a disubstituted amine.

[0579] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -Z3-X-[payload]; or [Payload]-X-Z1-(Aa) m -Z2-(Aa) n (Lys)-(Aa) o -N-((CH2)1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; or ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[Payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, n, o, p, and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxylic acid linker, and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0580] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) n +(Aa) o is >0.

[0581] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the structure: ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[payload]; or [Payload]-X-(Aa)m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2; or ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, p, and p* are integers ranging from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m Or the N-terminus of the disubstituted amine (N) is (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and X is absent or consists of or comprises a self-immolative group, preferably PABC.

[0582] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) n -(Lys)-(Aa) o contains the sequence motif Arg-Lys (RK) or His-Lys (HK) (N->C orientation).

[0583] In another embodiment, the present invention relates to a linker according to the present invention, comprising (Aa) n-(Lys)-(Aa) o is or includes RK or RKAA (N->C direction).

[0584] In another embodiment, the present invention relates to a linker according to the present invention, wherein the linker has the following structure: [ka] It comprises or consists of:

[0585] With regard to the exact composition of the linker and preferred embodiments thereof, the same applies mutatis mutandis as described above in the context of the ADCs of the invention.

[0586] Another aspect of the invention relates to methods for the preparation of ADCs according to the invention. The ADCs may be prepared using the methods disclosed in WO 2023 / 161291, which is incorporated herein by reference in its entirety.

[0587] In a particular embodiment, the present invention relates to a method for the preparation of an antibody-drug conjugate, comprising the step of conjugating a peptide linker according to the present invention to an antibody.

[0588] That is, any of the peptide linkers comprising at least a first and a second topoisomerase I inhibitor as disclosed herein can be conjugated to an antibody. In particular, any of the amine-containing peptide linkers disclosed herein can be conjugated to a glutamine residue of an antibody via transglutaminase. As disclosed elsewhere herein, the glutamine residue to which the peptide linker is conjugated may be an endogenous glutamine residue (e.g., Q295 of an IgG antibody) or a glutamine residue that has been introduced into the antibody by molecular engineering.

[0589] In a particular embodiment, the present invention relates to a method for the conjugation of a peptide linker according to the present invention using transglutaminase (TG), comprising: a) mixing an antibody, a peptide linker and transglutaminase (TG) in a fluid, thereby conjugating the linker-payload to the antibody in one step under the catalytic effect of the TG; and b) extracting the conjugate obtained in step a) from the fluid.

[0590] Therefore, the present invention further encompasses a method for conjugating a peptide linker according to the present invention to an antibody by transglutaminase in a one-step reaction. To this end, the antibody can be mixed with the peptide linker according to the present invention and transglutaminase in a fluid. A "fluid" within the meaning of the present invention is a liquid. Preferably, the liquid is an aqueous solution, even more preferably a buffered aqueous solution.

[0591] The peptide linker according to the present invention can be mixed with the antibody and transglutaminase by mixing a solution containing the peptide linker with a solution containing the antibody and a solution containing transglutaminase. Alternatively, solutions containing the peptide linker, antibody, and transglutaminase separately can be added to an aqueous solution. In particular, each component can be added to an aqueous solution at a defined concentration. The peptide linker according to the present invention is conjugated to the antibody under the catalytic effect of the transglutaminase. That is, the individual components can be mixed under conditions suitable for efficient conjugation of the peptide linker to the antibody. Such conditions are defined elsewhere herein.

[0592] In a second method step, the resulting antibody-payload conjugate must be removed from the liquid. Those skilled in the art are aware of methods for isolating antibody-payload conjugates from aqueous solutions. Furthermore, those skilled in the art are aware of methods for separating antibody-payload conjugates from unconjugated antibody or peptide linkers or from incompletely conjugated antibodies. For example, the antibody-payload conjugates according to the present invention can be isolated from the mixture by HPLC.

[0593] It should be understood that "extracting the conjugate from a fluid" is synonymous with "isolating the conjugate from a mixture," i.e., the conjugate can also be extracted by removing transglutaminase and the unconjugated antibody and peptide linker from the fluid.

[0594] The peptide linker used in the method according to the invention can be any one of the peptide linkers disclosed herein, in particular any peptide linker within the definition provided herein above or any peptide linker shown in the experimental examples.

[0595] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the peptide linker is a peptide linker of the invention.

[0596] Furthermore, the antibody may be as defined in more detail elsewhere herein, i.e., an antibody for an antibody-payload conjugate according to the present invention.

[0597] In particular, the peptide linker may comprise an amino acid sequence as set forth in SEQ ID NOs: 1 to 29 or 82 to 93. Furthermore, the linker may be any one of the linkers shown in Figures 4, 6, 7 or 8.

[0598] That is, in a particular embodiment, the present invention relates to a method according to the invention, wherein the peptide linker is conjugated to a glutamine residue contained in the antibody via a primary amine contained in an amino acid residue of the peptide linker.

[0599] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is an antibody fragment as defined elsewhere herein.

[0600] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is an IgA, IgD, IgE, IgG or IgM antibody.

[0601] In a particular embodiment, the invention relates to a method according to the invention, wherein the peptide linker is conjugated to a glutamine residue comprised in the Fc domain of the antibody.

[0602] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the glutamine residue to which the peptide linker is conjugated is the C of an IgG antibody. H The glutamine residue is Q295 (EU numbering) in the 2 domain.

[0603] In a particular embodiment, the invention relates to a method according to the invention, wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0604] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is a C of a non-glycosylated IgG antibody. H The domain is N297Q (EU numbering).

[0605] In a particular embodiment, the invention relates to a method according to the invention, wherein the glutamine residue introduced into the antibody heavy or light chain by molecular engineering is comprised in a peptide (a) incorporated into the antibody heavy or light chain or (b) fused to the N- or C-terminus of the antibody heavy or light chain.

[0606] In a particular embodiment, the invention relates to a method according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0607] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is a glycosylated IgG antibody.

[0608] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the IgG antibody is C H It is glycosylated at residue N297 (EU numbering) in the 2 domain.

[0609] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of m290, trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, loncastirucchii ... cimab, mecbotamab, adecatumumab, D93, gatipotuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab and endrecolomab; and / or The antibody specifically binds to an antigen selected from the group consisting of CD30, Her2 / neu, CD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, NaPi2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, Nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and Nectin-4.

[0610] In a particular embodiment, the invention relates to a method according to the invention, wherein the peptide linker is conjugated to the γ-carboxamide group of a Gln residue comprised in the antibody.

[0611] In certain embodiments, the invention relates to a method according to the invention, wherein the peptide linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0612] That is, in certain embodiments, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% efficiency. In a preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 70% efficiency. In another preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 75% efficiency. In another preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 80% efficiency. In another preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 85% efficiency. In another preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 90% efficiency. In another preferred embodiment, a peptide linker according to the present invention can be conjugated to a glycosylated antibody with at least 95% efficiency. Preferably, the glycosylated antibody is a glycosylated IgG antibody, more preferably an IgG antibody that is glycosylated at residue N297 (EU numbering).

[0613] Those skilled in the art will recognize methods for determining the conjugation efficiency of an antibody with a particular peptide linker. For example, conjugation efficiency can be determined as described herein. That is, an antibody, particularly an IgG1 antibody, can be incubated at a concentration of 1-5 mg / mL with 5-20 eq molar equivalents of linker and 3-6 U of microbial transglutaminase per mg of antibody in a suitable buffer at 37°C for 20-48 hours or as described in Example 1. After the incubation period, conjugation efficiency can be determined by LC-MS analysis under reducing conditions. The microbial transglutaminase can be, for example, MTG from Streptomyces mobaraensis, available from Zedira (Germany). Suitable buffers can be Tris, MOPS, HOPES, PBS, or bis-Tris buffer. However, it should be understood that the choice of buffer system can vary and may depend largely on the chemical properties of the linker. However, those skilled in the art will be able to identify optimal buffer conditions based on the present disclosure. Alternatively, conjugation efficiency may be determined as described by Spycher et al. (Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase, ChemBioChem 2019 18(19):1923-1927) or analyzed as in Benjamin et al. (Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering, Mol. Pharmaceutics 2019, 16:2795-2807).

[0614] In certain embodiments, antibodies can be conjugated by incubating 5 mg / ml of native glycosylated monoclonal antibody in 50 mM Tris pH 7.6 at 37°C for 24 hours in a rotating thermomixer with microbial transglutaminase (MTG, Zedira) at a concentration of 5-10 U / mg antibody and 5 molar equivalents of the indicated linker-payload. However, it should be understood that conditions, particularly buffer conditions and peptide linker concentration, can be adjusted depending on the characteristics of the payload. However, one of skill in the art will be able to identify optimal reaction conditions based on the teachings provided herein.

[0615] In a particular embodiment, the invention relates to a method according to the invention, wherein the transglutaminase is a microbial transglutaminase (MTG).

[0616] The transglutaminase for use in the methods of the invention can be any transglutaminase suitable for conjugating the peptide linkers of the invention to antibodies. The transglutaminase can be of any origin, for example, the transglutaminase can be of bacterial, archaeal or eukaryotic origin.

[0617] In certain embodiments, the transglutaminase can be a mammalian transglutaminase, including human transglutaminase, hi certain embodiments, the transglutaminase can be a microbial transglutaminase, including bacterial and fungal transglutaminase.

[0618] In a particular embodiment, the invention relates to a method according to the invention, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular from Streptomyces mobaraensis.

[0619] That is, the microbial transglutaminase used in the method of the present invention may be derived from Streptomyces species, particularly Streptomyces mobaraensis, and preferentially has 80% sequence identity to the native enzyme. Thus, MTG may be the native enzyme or an engineered variant of the native enzyme.

[0620] One such microbial transglutaminase is commercially available from Zedira (Germany). It is recombinantly produced in E. coli. Streptomyces mobaraensis transglutaminase has the amino acid sequence as disclosed in SEQ ID NO: 78. S. mobaraensis MTG variants with other amino acid sequences have been reported and are also encompassed by the present invention (SEQ ID NOs: 79 and 80).

[0621] One such microbial transglutaminase could also be the MTG-TX variant from S. mobaraensis described in Jin et al. 2016, Journal of Molecular Catalysis B:Enzymatic, which exhibits high salt tolerance and a wide range of pH and temperature stability.

[0622] In another embodiment, a microbial transglutaminase from Streptomyces ladakanum (formerly known as Streptoverticillium ladakanum) can be used. Streptomyces ladakanum transglutaminase (U.S. Pat. No. 6,660,510 B2) has the amino acid sequence as disclosed in SEQ ID NO:81.

[0623] Both of the above transglutaminases may be sequence modified. In some embodiments, a transglutaminase having 80%, 85%, 90%, or 95% or more sequence identity to any one of SEQ ID NOs: 78-81 may be used.

[0624] Another suitable microbial transglutaminase is commercially available from Ajinomoto and is called ACTIVA TG. Compared to the transglutaminase from Zedira, ACTIVA TG lacks four N-terminal amino acids but has similar activity.

[0625] Further microbial transglutaminases that may be used in the context of the present invention are disclosed in Kieliszek and Misiewicz (Folia Microbiol (Praha). 2014; 59(3): 241-250), WO 2015 / 191883 A1, WO 2008 / 102007 A1 and US 2010 / 0143970, the contents of which are incorporated herein by reference in their entirety.

[0626] In certain embodiments, mutant variants of microbial transglutaminase may be used for conjugating a linker to an antibody. That is, the microbial transglutaminase used in the methods of the invention may be a variant of S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 or 79. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutation G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutations G254D and E304D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutations D8E and G254D. In certain embodiments, a recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutations E124A and G254D. In certain embodiments, a recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutations A216D and G254D. In certain embodiments, a recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 may comprise the mutations G254D and K331T.

[0627] In a particular embodiment, the invention relates to a method according to the invention, wherein transglutaminase is added to the conjugation reaction at a concentration of less than 200 U / mg antibody.

[0628] Microbial transglutaminase can be added to the conjugation reaction at any concentration that allows for efficient conjugation of the linker and the antibody. In certain embodiments, the concentration of microbial transglutaminase in the conjugation reaction can depend on the amount of antibody used in the same reaction. For example, microbial transglutaminase can be added to the conjugation reaction at a concentration of less than 200 U / mg antibody, 150 U / mg antibody, 100 U / mg antibody, 90 U / mg antibody, 80 U / mg antibody, 70 U / mg antibody, 60 U / mg antibody, 50 U / mg antibody, 40 U / mg antibody, 30 U / mg antibody, 20 U / mg antibody, 10 U / mg antibody, or 6 U / mg antibody.

[0629] In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 3 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 5 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 6 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 7.5 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 10 U / mg antibody.

[0630] In certain embodiments, the microbial transglutaminase may be added to the conjugation reaction at a concentration of 1-100 U / mg antibody. In certain embodiments, the microbial transglutaminase may be added to the conjugation reaction at a concentration of 3-50 U / mg antibody. In certain embodiments, the microbial transglutaminase may be added to the conjugation reaction at a concentration of 5-25 U / mg antibody.

[0631] In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1-20 U / mg antibody, preferably 3-15 U / mg antibody, and more preferably 5-10 U / mg antibody.

[0632] Preferably, the transglutaminase used in the method of the present invention is a microbial transglutaminase.However, it should be noted that the equivalent reaction can be carried out by an enzyme containing transglutaminase activity of non-microbial origin.Therefore, the antibody-payload conjugate according to the present invention can also be produced by an enzyme containing transglutaminase activity of non-microbial origin.

[0633] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is added to the conjugation reaction at a concentration of 0.1 to 50 mg / mL.

[0634] The antibody can be added to the conjugation reaction at any concentration suitable for efficient antibody conjugation. However, it is preferred that the antibody be added to the conjugation reaction at a concentration in the range of 0.1 to 50 mg / mL. That is, in certain embodiments, the present invention relates to methods according to the present invention, in which the antibody is added to the conjugation reaction at a concentration of 0.1 to 50 mg / mL, preferably 0.25 to 25 mg / mL, more preferably 0.5 to 12.5 mg / mL, even more preferably 1 to 10 mg / mL, even more preferably 2 to 7.5 mg / mL, and most preferably about 5 mg / mL.

[0635] Alternatively, the antibody may be added to the conjugation reaction at a concentration ranging from 1 to 20 mg / ml, preferably from 2.5 to 20 mg / mL, more preferably from 5 to 20 mg / mL, and most preferably from 5 to 17 mg / mL.

[0636] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 100 molar equivalents of the peptide linker.

[0637] To obtain efficient conjugation, the linker is preferably added in molar excess to the antibody, i.e., in certain embodiments, the antibody is mixed with at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 molar equivalents of linker.

[0638] That is, in a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 100 molar equivalents of a linker, preferably 2 to 80 molar equivalents of a linker, more preferably 2 to 70 molar equivalents of a linker, even more preferably 2 to 60 molar equivalents of a linker, even more preferably 2 to 50 molar equivalents of a linker, even more preferably 2 to 40 molar equivalents of a linker, even more preferably 2 to 30 molar equivalents of a linker, even more preferably 2 to 25 molar equivalents of a linker, even more preferably 2 to 20 molar equivalents of a linker, even more preferably 2 to 15 molar equivalents of a linker, and most preferably 2 to 10 molar equivalents of a linker.

[0639] Alternatively, the antibody may be contacted with 2.5 to 100 molar equivalents of the linker, preferably 2.5 to 80 molar equivalents of the linker, more preferably 2.5 to 70 molar equivalents of the linker, even more preferably 2.5 to 60 molar equivalents of the linker, even more preferably 2.5 to 50 molar equivalents of the linker, even more preferably 2.5 to 40 molar equivalents of the linker, even more preferably 2.5 to 30 molar equivalents of the linker, even more preferably 2.5 to 20 molar equivalents of the linker, even more preferably 2.5 to 15 molar equivalents of the linker, even more preferably 2.5 to 10 molar equivalents of the linker, and most preferably 2.5 to 8 molar equivalents of the linker.

[0640] Alternatively, the antibody may be contacted with 5 to 100 molar equivalents of the linker, preferably 5 to 80 molar equivalents of the linker, more preferably 5 to 70 molar equivalents of the linker, even more preferably 5 to 60 molar equivalents of the linker, even more preferably 5 to 50 molar equivalents of the linker, even more preferably 5 to 40 molar equivalents of the linker, even more preferably 5 to 30 molar equivalents of the linker, even more preferably 5 to 20 molar equivalents of the linker, even more preferably 5 to 15 molar equivalents of the linker, and most preferably 5 to 10 molar equivalents of the linker.

[0641] In a particular embodiment, the invention relates to a method according to the invention, wherein the conjugation reaction may be carried out in a buffered solution.

[0642] The method according to the invention is preferably carried out at a pH in the range of 5 to 10. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at a pH in the range of 5 to 10, preferably in the range of 6 to 9, more preferably in the range of 6 to 8.5, even more preferably in the range of 6.5 to 8, and most preferably in the range of 6.6 to 7.6.

[0643] In certain embodiments, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at pH 6.6.

[0644] In certain embodiments, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at pH 7.6.

[0645] The methods of the present invention can be carried out in any buffer suitable for conjugating a payload to a linker. Suitable buffers for the methods of the present invention include, but are not limited to, Tris, MOPS, HEPES, PBS, or Bis-Tris buffer. The buffer concentration depends, inter alia, on the concentration of the antibody and / or linker and can range from 10 to 1000 mM, 10 to 500 mM, 10 to 400 mM, 10 to 250 mM, 10 to 150 mM, or 10 to 100 mM. Furthermore, the buffer can contain any salt concentration suitable for carrying out the methods of the present invention. For example, buffers used in the methods of the invention may have a salt concentration of ≦250 mM, ≦200 mM, ≦150 mM, ≦140 mM, ≦130 mM, ≦120 mM, ≦110 mM, ≦100 mM, ≦90 mM, ≦80 mM, ≦70 mM, ≦60 mM, ≦50 mM, ≦40 mM, ≦30 mM, ≦20 mM or ≦10 mM, or may have no salt.

[0646] That is, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the buffer solution comprises: a) a pH in the range of 5 to 10; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) salt concentration less than 250 mM Includes:

[0647] In a preferred embodiment, the present invention relates to a method according to the invention, wherein the buffer solution comprises: a) a pH in the range of 6 to 9; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) salt concentration less than 250 mM Includes:

[0648] In a more preferred embodiment, the present invention relates to a method according to the invention, wherein the buffer solution comprises: a) a pH in the range of 6 to 8; and / or b) a buffer concentration in the range of 10 to 500 mM; and / or c) salt concentration less than 150 mM Includes:

[0649] In an even more preferred embodiment, the present invention relates to a method according to the invention, wherein the buffer solution comprises a) a pH in the range of 6 to 8; and / or b) a buffer concentration in the range of 10 to 200 mM; and / or c) salt concentration less than 50 mM Includes:

[0650] In a preferred embodiment, the method of the present invention is carried out in 50 mM Tris (pH 7.6), preferably without salt.

[0651] In another preferred embodiment, the method of the present invention is carried out in 50 mM BisTris (pH 6.6), preferably without salt.

[0652] In another preferred embodiment, the method of the present invention is carried out in 50 mM BisTris (pH 7.5), preferably without salt.

[0653] It should be noted that optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary between payloads and may depend to some extent on the physicochemical properties of the linker and / or payload, however, one of ordinary skill in the art would not require undue experimentation to identify suitable reaction conditions for carrying out the methods of the present invention.

[0654] It should be understood that the present application encompasses any combination of linkers, antibodies, MTG and / or buffer concentrations disclosed above.

[0655] In certain embodiments, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 80 molar equivalents of a linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 20 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 0.1 to 20 mg / mL.

[0656] In a preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 50 molar equivalents of a linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 1 to 20 mg / mL.

[0657] In a more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 30 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 2.5 to 20 mg / mL.

[0658] In an even more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 20 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 2.5 to 20 mg / mL.

[0659] In an even more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 15 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 5 to 20 mg / mL.

[0660] In a most preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2.5 to 12.5 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 5 to 20 mg / mL.

[0661] In another preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 20 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 2.5 to 20 mg / mL.

[0662] It should be noted that the specific reaction mixtures disclosed above can be freely combined with any of the buffer conditions disclosed herein, however, it is preferred that the specific components as defined above are mixed at a pH in the range of 6-8.

[0663] In certain embodiments, the present invention relates to an antibody-payload conjugate produced by a method according to the present invention.

[0664] That is, the present invention further relates to an antibody-linker conjugate produced by any of the aforementioned method steps.

[0665] In the present invention, the subject is, in preferred embodiments, a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee, and human. In the most preferred embodiment, the subject is a human.

[0666] Other aspects and advantages of the present invention will be described in the following examples, which are given for purposes of illustration and not limitation. Each article, patent, patent application, or other document cited in this application is incorporated herein by reference in its entirety.

[0667] List of embodiments: P1. A compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being As a first payload, a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and An antibody-drug conjugate (ADC) having as a second payload a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable. P2. The ADC according to embodiment 1, wherein the linker is a peptide linker. P3. The first payload and / or the second payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue contained in the peptide linker; preferably, 3. The ADC according to embodiment 1 or 2, wherein the first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa. P4. The linker has the following structure (N->C direction): [Payload 1]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 2]; or [Payload 2]-XZ-(Aa)mZ-(Aa) n (Lys)-(Aa)oZX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z is absent or is a spacer comprising an alkyl or heteroalkyl group, preferably the spacer is (CH2)2; and The ADC according to any one of embodiments 1 to 3, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. P5. The ADC according to any one of embodiments 1-4, wherein the ADC comprises two or more first payloads and / or two or more second payloads. P6. An ADC according to any one of embodiments 1-5, wherein the camptothecin is exatecan or deruxtecan. P7. The ADC according to any one of embodiments 1-6, wherein the second payload has a glycine residue linked to said topoisomerase I inhibitor of the second payload. P8. The linker has the following structure (N->C direction): [ka] 8. An ADC according to any one of embodiments 1 to 7, comprising or consisting of: P9. Antibodies include trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamb, and adecatum and / or selected from the group consisting of mab, D93, gatipotuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab and endrecolomab; and / or Antibodies include CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, and NaP. 9. The ADC according to any one of embodiments 1 to 8, which specifically binds to an antigen selected from the group consisting of i2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, Nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and Nectin-4. P10. The ADC according to any one of embodiments 1-9, wherein said ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"). P11. The ADC according to any one of embodiments 1-9, wherein said linker further comprises a third payload, preferably a toxin or cytotoxin, more preferably an auristatin, even more preferably MMAE (monomethylauristatin E). P12. A pharmaceutical composition comprising an ADC according to any one of embodiments 1-11 and at least one pharmaceutically acceptable ingredient. P13. An ADC according to any one of embodiments 1-11 for use in a method for treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic disease. P14. the antibody-payload conjugate comprises trastuzumab, and the neoplastic disease is HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer; the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell related cancer; preferably, the B-cell related cancer is non-Hodgkin's lymphoma, in particular, the B-cell related cancer is diffuse large B-cell lymphoma; or The ADC according to any one of embodiments 1 to 11 and 13 or the pharmaceutical composition of embodiment 12, wherein the antibody-payload conjugate comprises enfortumab or an enfortumab variant, and the neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer. P15. A compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being As a first payload, a cell membrane-permeable camptothecin cytotoxic molecule; and as a second payload, a camptothecin cytotoxic molecule that is not cell membrane permeable; the cytotoxic molecules of the first and second payloads are exatecan; the second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; the ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4 "DAR4"); and The antibody is an IgG, preferably an IgG1 antibody. P16. The ADC according to embodiment 15, wherein the antibody is trastuzumab.

[0668] 1. A compound of formula AL, wherein A is an antibody or antibody fragment and L is a linker, said linker being: As a first payload, a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and An antibody-drug conjugate (ADC) having as a second payload a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable. 2. An ADC according to embodiment 1, wherein the camptothecin is exatecan or deruxtecan. 3. The ADC according to embodiment 1 or 2, wherein the second payload is modified to reduce its cell membrane permeability. 4. The ADC according to any one of embodiments 1-3, wherein the second payload has a glycine residue linked to said topoisomerase I inhibitor of the second payload. 5. The ADC according to any one of embodiments 1-4, wherein the linker is a peptide linker. 6. The first payload and / or the second payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue contained in the peptide linker; preferably, 6. The ADC according to embodiment 5, wherein the first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa. 7. The linker is conjugated to the antibody via an isopeptide bond formed between a glutamine residue in the antibody and a primary amine in the linker, preferably the primary amine is contained in a lysine residue, lysine mimetic, or lysine derivative in the peptide linker, or the primary amine is contained in a peptide linker having the structure NH2-(CH2) 1~10 7. The ADC according to any of embodiments 1-6, comprising an amino acid residue having a -COOH. 8. The ADC according to any one of embodiments 1-7, wherein the linker further comprises at least one positively charged amino acid residue, preferably, the at least one positively charged amino acid residue is selected from arginine and / or histidine. 9. The linker has the following structure: [Payload 1]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 2]; or [Payload 2]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2; and 9. The ADC according to any one of embodiments 1 to 8, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. 10.(Aa) m +(Aa) n +(Aa) o is >0, preferably (Aa) n +(Aa) o 10. The ADC according to embodiment 9, wherein: 11.Z2 is (Aa) m The N-terminus of (Aa) n or (Lys) is a dicarboxylic acid linked to the N-terminus of (Aa); and one payload is directly or indirectly linked to (Aa) m and the other payload is directly or indirectly linked to (Lys) or (Aa) o or vice versa. 12. The linker has the following structure: [Payload 1]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[Payload 2]; or [Payload 2]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa)oX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer ranging from 1 to 10, preferably from 1 to 6, more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m The N-terminus of (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and 12. The ADC according to any one of embodiments 9-11, wherein X consists of or comprises an absent or self-immolative group, preferably PABC. 13.(Aa) n -(Lys)-(Aa) o 13. The ADC according to any one of embodiments 9 to 12, wherein said amino acid sequence is Arg-Lys(RK) or His-Lys(HK) (N->C orientation). 14.(Aa) n -(Lys)-(Aa) o is or comprises RK or RKAA (N→C direction). 15. The linker has the following structure: [ka] 15. An ADC according to any one of embodiments 1-14, comprising or consisting of: 16. The ADC according to any one of embodiments 1 to 15, wherein the ADC comprises two or more first payloads and / or two or more second payloads. 17. The ADC according to any one of embodiments 1-16, wherein said ADC consists of two first payloads and two second payloads (drug-to-antibody ratio of 4, "DAR4"). 18. The ADC according to any one of embodiments 1-16, wherein said linker further comprises a third payload, preferably a toxin or cytotoxin, more preferably an auristatin, even more preferably MMAE (monomethylauristatin E). 19. The ADC according to embodiment 18, wherein the linker is a peptide linker and the third payload is linked to the N- or C-terminus of the peptide linker, or the third payload is linked to a side chain of an amino acid residue comprised in the peptide linker. 20. An ADC according to embodiment 19, wherein the two payloads are linked to the same functional group of the peptide linker, preferably via a chemical linker comprising a disubstituted amine. 21. The linker has the structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -Z3-X-[payload]; or [Payload]-X-Z1-(Aa) m -Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; or ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[Payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, n, o, p, and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxylic acid linker, and 21. The ADC according to embodiment 20, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. 22.(Aa) n +(Aa) o 22. The ADC according to embodiment 21, wherein: 23. The linker has the structure: ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[payload]; or [Payload]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2; or ([Payload]-X-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -X-[payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, p, and p* are integers ranging from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m Or the N-terminus of the disubstituted amine (N) is (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and 23. The ADC according to any one of embodiments 20-22, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. 24.(Aa) n -(Lys)-(Aa) o comprises the sequence motif Arg-Lys(RK) or His-Lys(HK) (N->C orientation). 25.(Aa) n -(Lys)-(Aa) o is or comprises RK or RKAA (N→C direction). 26. The linker has the following structure: [ka] 26. An ADC according to any one of embodiments 18 to 25, comprising or consisting of: 27. The ADC according to any one of embodiments 1-26, wherein the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody. 28. Glutamine residues are the C H 28. The ADC according to any one of embodiments 7-27, wherein the PG is residue Q295 (EU numbering) of the PG-2 domain. 29. The ADC according to any one of embodiments 1-28, wherein the antibody is a glycosylated antibody, preferably wherein the antibody is an IgG antibody glycosylated at residue N297 (EU numbering). 30. Antibodies include trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamb, and adecatum and / or selected from the group consisting of mab, D93, gatipotuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab and endrecolomab; and / or Antibodies include CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, and NaP. 30. The ADC according to any one of embodiments 1 to 29, which specifically binds to an antigen selected from the group consisting of i2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, Nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and Nectin-4. 31. A pharmaceutical composition comprising an ADC according to any one of embodiments 1 to 30 and at least one pharmaceutically acceptable ingredient. 32. An ADC according to any one of embodiments 1 to 30 or the pharmaceutical composition of claim 28 for use in a method for treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic disease (particularly, the neoplastic disease is cancer). 33. the antibody-payload conjugate comprises trastuzumab, and the neoplastic disease is HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer; the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell related cancer; preferably, the B-cell related cancer is non-Hodgkin's lymphoma, in particular, the B-cell related cancer is diffuse large B-cell lymphoma; or The ADC according to any one of embodiments 1 to 30 and 32 or the pharmaceutical composition of embodiment 31 or 32, wherein the antibody-payload conjugate comprises enfortumab or an enfortumab variant, and the neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer. 34. the antibody-payload conjugate comprises an antibody targeting NaPi2b, preferably the antibody targeting NaPi2b is upifitamab having a heavy chain as set forth in SEQ ID NO: 98 and a light chain as set forth in SEQ ID NO: 99, or a heavy chain as set forth in SEQ ID NO: 100 and a light chain as set forth in SEQ ID NO: 101, and the neoplastic disease is a NaPi2b-positive cancer, in particular a NaPi2b-positive lung cancer or ovarian cancer; Preferably, the ADC has the structure: [ka] 33. An ADC according to any one of embodiments 1 to 30 and 32 or the pharmaceutical composition of embodiment 31 or 32, comprising a linker having the formula: 35. the antibody-payload conjugate comprises an antibody targeting Nectin-4, preferably the antibody targeting Nectin-4 is m290 having a heavy chain as set forth in SEQ ID NO: 96 and a light chain as set forth in SEQ ID NO: 97, or enfortumab having a heavy chain as set forth in SEQ ID NO: 75 or 94 and a light chain as set forth in SEQ ID NO: 95, 76 or 77, and the neoplastic disease is Nectin-4 positive cancer, in particular Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer; Preferably, the ADC has the structure: [ka] 33. An ADC according to any one of embodiments 1 to 30 and 32 or the pharmaceutical composition of embodiment 31 or 32, comprising a linker having the formula: 36. A linker comprising a first and a second payload, wherein the first payload is a cell membrane permeable topoisomerase I inhibitor, preferably a cell membrane permeable camptothecin cytotoxic molecule; and the second payload is a topoisomerase I inhibitor that is not cell membrane permeable, preferably a camptothecin cytotoxic molecule that is not cell membrane permeable; Preferably, the linker is for conjugation to an antibody. 37. The linker according to embodiment 36, wherein the camptothecin is exatecan or deruxtecan. 38. The linker according to embodiment 36 or 37, wherein the second payload is modified to reduce its cell membrane permeability. 39. A linker according to any one of embodiments 36-38, wherein the second payload has a glycine residue linked to said topoisomerase I inhibitor of the second payload. 40. A linker according to any one of embodiments 36-39, wherein the linker is a peptide linker. 41. The first payload and / or the second payload are linked to the N- or C-terminus of the peptide linker or to the side chain of an amino acid residue contained in the peptide linker; preferably, 41. The linker according to embodiment 40, wherein the first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa. 42. The linker comprises a primary amine for conjugation to an antibody, preferably the primary amine is contained in a lysine residue, a lysine mimetic, or a lysine derivative; or the primary amine has the structure NH2-(CH2) 1~10 A linker according to any of embodiments 36 to 41, which is comprised in an amino acid residue having a —COOH; preferably, the amino acid residue is comprised in the peptide linker. 43. A linker according to any one of embodiments 36 to 42, wherein the linker further comprises at least one positively charged amino acid residue, preferably wherein the at least one positively charged amino acid residue is selected from arginine and / or histidine. 44. The linker has the following structure: [Payload 1]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 2]; or [Payload 2]-X-Z1-(Aa) m -Z2-(Aa) n -(Lys)-(Aa) o -Z3-X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2; and 44. A linker according to any one of embodiments 36 to 43, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. 45.(Aa) m +(Aa) n +(Aa) o is >0, preferably (Aa) n +(Aa) o 45. A linker according to embodiment 44, wherein: 46.Z2 is (Aa) m The N-terminus of (Aa) nor (Lys) is a dicarboxylic acid linked to the N-terminus of (Aa); and one payload is directly or indirectly linked to (Aa) m and the other payload is directly or indirectly linked to (Lys) or (Aa) o or vice versa. 47. The linker has the following structure: [Payload 1]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa) o -X-[Payload 2]; or [Payload 2]-X-(Aa) m -(dicarboxylic acid)-(Aa) n (Lys)-(Aa)oX-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer ranging from 1 to 10, preferably from 1 to 6, more preferably from 1 to 4; n and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4, and n+o is >0; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; (Dicarboxylic acid) is (Aa) m The N-terminus of (Aa) n or a dicarboxylic acid linked to the N-terminus of (Lys); and 47. A linker according to any one of embodiments 44 to 46, wherein X is absent or consists of or comprises a self-immolative group, preferably PABC. 48.(Aa) n -(Lys)-(Aa) o 48. A linker according to any one of embodiments 44 to 47, wherein said linker comprises the sequence motif Arg-Lys(RK) or His-Lys(HK) (N->C orientation). 49.(Aa) n-(Lys)-(Aa) o is or comprises RK or RKAA (N->C orientation). 50. The linker has the following structure: [ka] 50. A linker according to any one of embodiments 36 to 49, comprising or consisting of: 51. A linker according to any one of embodiments 36 to 50, wherein said linker further comprises a third payload, preferably a toxin or cytotoxin, more preferably an auristatin, even more preferably MMAE (monomethylauristatin E). 52. A linker according to embodiment 51, wherein the linker is a peptide linker and the third payload is linked to the N- or C-terminus of the peptide linker, or the third payload is linked to the side chain of an amino acid residue contained in the peptide linker. 53. A linker according to embodiment 52, wherein the two payloads are linked to the same functional group of the peptide linker, preferably via a chemical linker comprising a disubstituted amine. 54. The linker has the structure: ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -Z3-X-[payload]; or [Payload]-X-Z1-(Aa) m -Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6 -C(=O)-(Aa) p / p* -Z3-X-[payload])2; or ([Payload]-X-Z1-(Aa) m / m* -C(=O)-(CH2) 1~6 )2-N-Z2-(Aa) n (Lys)-(Aa) o -N-((CH2) 1~6-C(=O)-(Aa) p / p* -Z3-X-[Payload])2 (In the formula, [payload] is a payload independently selected from a first payload, a second payload, and a third payload, and the linker comprises all three payloads; (Aa) is any amino acid residue; m, m*, n, o, p, and p* may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative; Z 1~3 is a spacer that is absent or comprises an alkyl or heteroalkyl group, preferably the spacer comprises (CH2)2, even more preferably Z2 is a dicarboxy...

Claims

1. Formula A-L (wherein A is an antibody or antibody fragment, L is a linker, and the linker is The first payload is a cell membrane-permeable topoisomerase I inhibitor; and The second payload is an antibody-drug conjugate (ADC) containing a non-cell membrane-permeable topoisomerase I inhibitor.

2. The ADC according to claim 1, wherein the topoisomerase I inhibitor is camptothecin.

3. The ADC according to claim 2, wherein the camptothecin is exatecan.

4. The ADC according to claim 1 or 2, wherein the second payload is modified to reduce its cell membrane permeability.

5. The ADC according to claim 1 or 2, wherein the second payload has a glycine residue linked to the topoisomerase I inhibitor of the second payload.

6. The ADC according to claim 1 or 2, wherein the linker is a peptide linker.

7. The ADC according to claim 6, wherein the first payload and / or the second payload are linked to the N or C terminus of the peptide linker or to the side chain of an amino acid residue contained in the peptide linker.

8. The ADC according to claim 7, wherein the first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa.

9. The linker is conjugated to the antibody via an isopeptide bond formed between a glutamine residue contained in the antibody and a primary amine contained in the linker, wherein the primary amine is contained in a lysine residue, lysine mimetic, or lysine derivative contained in the peptide linker, or the primary amine is contained in a structure NH contained in the peptide linker. ２ - (CH ２ ) １～１０ The ADC according to claim 1 or 2, which is contained in an amino acid residue having -COOH.

10. The ADC according to claim 1 or 2, wherein the linker further comprises at least one positively charged amino acid residue selected from arginine and / or histidine.

11. The linker has the following structure: [Payload 1] - X - Z １ - (Aa) ｍ - Z ２ - (Aa) ｎ - (Lys) - (Aa) ｏ - Z ３ - X - [Payload 2]; or [Payload 2] -X-Z １ - (Aa) ｍ -Z ２ - (Aa) ｎ -(Lys)-(Aa) ｏ -Z ３ -X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o are integers in the range of 0 to 10; (Lys) is a lysine residue, lysine mimetic, or lysine derivative; Z １～３ This is a spacer that is either absent or contains an alkyl or heteroalkyl group; and The ADC according to claim 1 or 2, wherein X is either absent or a self-sacrificing group (or comprises or includes the ADC).

12. (Aa) ｎ + (Aa) ｏ However, the ADC according to claim 11, wherein > 0.

13. Z ２ However, (Aa) ｍ The N-terminus of (Aa) ｎ or a dicarboxylic acid linked to the N-terminus of (Lys); and one of the payloads is directly or indirectly (Aa) ｍ The C-terminus of is connected, and the other payload is directly or indirectly connected to (Lys) or (Aa) ｏ The ADC according to claim 11, wherein it is connected to the C-terminus of or vice versa.

14. The linker has the following structure: [Payload 1] -X-(Aa) ｍ -(dicarboxylic acid)-(Aa) ｎ (Lys)-(Aa) ｏ -X-[Payload 2]; or [Payload 2] -X-(Aa) ｍ -(dicarboxylic acid)-(Aa) ｎ (Lys)-(Aa)o-X-[payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer in the range of 1 to 10; n and o are integers in the range of 0 to 10, and n + o is > 0; (Lys) is a lysine residue, lysine mimetic, or lysine derivative; (Dicarboxylic acid) is (Aa) ｍ The N-terminus of (Aa) ｎ or a dicarboxylic acid linked to the N-terminus of (Lys); and The ADC according to claim 11, wherein X is either absent or a self-sacrificing group (or a self-sacrificing group).

15. m is an integer in the range of 2 to 4; and / or n and o are integers in the range of 0 to 4; and / or The ADC according to claim 14, wherein X is PABC.

16. (Aa) ｎ -(Lys)-(Aa) ｏ but includes an array motif Arg-Lys (RK) or His-Lys (HK) (N->C direction); and / or (Aa) ｎ -(Lys)-(Aa) ｏ The ADC according to claim 11, wherein the ADC is RK or RKAA (N->C direction) or includes the same.

17. The linker has the following structure: 【Chemistry 1】 The ADC according to claim 1, comprising or consisting of the following.

18. The ADC includes two or more first payloads and / or two or more second payloads; and / or The ADC according to claim 1 or 2, wherein the ADC comprises two first payloads and two second payloads (drug-to-antibody ratio "DAR4").

19. The ADC according to claim 1 or 2, wherein the antibody is an IgG antibody.

20. The aforementioned glutamine residue is C of the IgG antibody. Ｈ The ADC according to claim 9, wherein the residue Q295 (EU numbering) is a two-domain residue.

21. The ADC according to claim 1 or 2, wherein the antibody is a glycosylated antibody, and the antibody is an IgG antibody glycosylated at residue N297 (EU numbering).

22. The aforementioned antibodies are trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, verantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, roncustuximab, mekubotamab, Selected from the group consisting of adecatumumab, D93, gatipotuzumab, rabetuzumab, tusamitamab, rifastuzumab, milbetuximab, sofituzumab, anetuzumab, tisotuzumab, cofituzumab, praluzatamab, radriatuzumab, verantamab, patrizumab, cetuximab, nimotuzumab, matsuzumab, portuzumab, sitatuzumab, tucotsuzumab, and endrecolomab; and / or The ADC according to claim 1 or 2, wherein the antibody specifically binds to an antigen selected from the group consisting of CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, NaPi2b, FRα, MUC16, mesoserine, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1.

23. A pharmaceutical composition comprising the ADC described in claim 1 or 2 and at least one pharmaceutically acceptable component.

24. The pharmaceutical composition according to claim 23 for use in a method of treating a patient who has a neoplastic disease, is at risk of developing it, and / or has been diagnosed with it.

25. The antibody-payload conjugate comprises trastuzumab, and the neoplastic disease is HER2-positive cancer; or The antibody-payload conjugate comprises polatuzumab, and the neoplastic disease is B-cell related cancer; or The pharmaceutical composition for use according to claim 24, wherein the antibody-payload conjugate comprises enfortumab or an enfortumab variant, and the neoplastic disease is nectin-4 positive cancer.

26. Whether the HER2-positive cancer is HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer; The B-cell-related cancer is non-Hodgkin lymphoma or diffuse large B-cell lymphoma; or The pharmaceutical composition for use according to claim 25, wherein the nectin-4 positive cancer is nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

27. The pharmaceutical composition for use according to claim 24, wherein the antibody-payload conjugate comprises an antibody that targets NaPi2b, and the neoplastic disease is NaPi2b-positive cancer.

28. A linker for conjugation to an antibody, comprising first and second payloads, wherein the first payload is a cell membrane-permeable topoisomerase I inhibitor; and The linker wherein the second payload is a topoisomerase I inhibitor that is not permeable to the cell membrane.

29. The linker according to claim 28, wherein the topoisomerase I inhibitor is camptothecin.

30. The linker according to claim 29, wherein the camptothecin is exatecan or deruxtecan.

31. The linker according to claim 28 or 29, wherein the second payload is modified to reduce its cell membrane permeability.

32. The linker according to claim 28 or 29, wherein the second payload has a glycine residue linked to the topoisomerase I inhibitor of the second payload.

33. The linker according to claim 28 or 29, wherein the linker is a peptide linker.

34. The linker according to claim 33, wherein the first payload and / or the second payload are linked to the N or C terminus of the peptide linker or to the side chain of an amino acid residue contained in the peptide linker.

35. The linker according to claim 34, wherein the first payload is linked to the N-terminus of the peptide linker and the second payload is linked to the C-terminus of the peptide linker, or vice versa.

36. The linker contains a primary amine for conjugation to an antibody, wherein the primary amine is contained in a lysine residue, a lysine mimetic, or a lysine derivative; or the primary amine is structurally NH ２ - (CH ２ ) １～１０ A linker according to claim 28 or 29, comprising an amino acid residue having a -COOH group.

37. The linker according to claim 36, wherein the amino acid residue is contained in the peptide linker.

38. The linker according to claim 28 or 29, wherein the linker further comprises at least one positively charged amino acid residue, the at least one positively charged amino acid residue being selected from arginine and / or histidine.

39. The linker has the following structure: [Payload 1] -X-Z １ - (Aa) ｍ -Z ２ - (Aa) ｎ -(Lys)-(Aa) ｏ -Z ３ -X-[Payload 2]; or [Payload 2] -X-Z １ - (Aa) ｍ -Z ２ - (Aa) ｎ -(Lys)-(Aa) ｏ -Z ３ -X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m, n, and o may be integers in the range of 0 to 10; (Lys) is a lysine residue, lysine mimetic, or lysine derivative; Z １～３ This is a spacer that is either absent or contains an alkyl or heteroalkyl group; and The linker according to claim 28 or 29, wherein X is either nonexistent or a self-sacrificing group (or comprising X).

40. (Aa) ｎ + (Aa) ｏ However, the linker according to claim 39, wherein > 0.

41. Z ２ However, (Aa) ｍ The N-terminus of (Aa) ｎ or a dicarboxylic acid linked to the N-terminus of (Lys); and one of the payloads is directly or indirectly (Aa) ｍ The C-terminus of is connected, and the other payload is directly or indirectly connected to (Lys) or (Aa) ｏ The linker according to claim 39, which is connected to the C-terminus of or vice versa.

42. The linker has the following structure: [Payload 1] -X-(Aa) ｍ -(dicarboxylic acid)-(Aa) ｎ (Lys)-(Aa) ｏ -X-[Payload 2]; or [Payload 2] -X-(Aa) ｍ -(dicarboxylic acid)-(Aa) ｎ (Lys)-(Aa) ｏ -X-[Payload 1]; (In the formula, [Payload 1] is the first payload; [Payload 2] is the second payload; (Aa) is any amino acid residue; m is an integer in the range of 1 to 10; n and o may be integers in the range of 0 to 10; (Lys) is a lysine residue, lysine mimetic, or lysine derivative; (Dicarboxylic acid) is (Aa) ｍ The N-terminus of (Aa) ｎ or a dicarboxylic acid linked to the N-terminus of (Lys); and The linker according to claim 39, wherein X is either nonexistent or a self-sacrificing group (or comprising X).

43. m is an integer in the range of 2 to 4; and / or n and o are integers in the range of 0 to 4; and / or The linker according to claim 42, wherein X is PABC.

44. (Aa) ｎ -(Lys)-(Aa) ｏ but includes an array motif Arg-Lys (RK) or His-Lys (HK) (N->C direction); and / or (Aa) ｎ -(Lys)-(Aa) ｏ The linker according to claim 39, wherein it is RK or RKAA (N->C direction) or includes the same.

45. The linker has the following structure: 【Chemistry 2】 The linker according to claim 28, comprising or consisting of the following.

46. A method for preparing an antibody-drug conjugate, comprising the step of conjugating an antibody with the peptide linker described in claim 28 or 29.

47. A method for conjugating a peptide linker according to claim 28 or 29, using transglutaminase (TG), comprising: a) mixing the antibody, the peptide linker, and transglutaminase (TG) in a fluid to conjugate the linker-payload to the antibody in one step under the catalytic effect of the TG; and b) extracting the conjugate obtained in step a) from the fluid.

48. The method according to claim 47, wherein the peptide linker is conjugated to a glutamine residue contained in the antibody via a primary amine contained in an amino acid residue of the peptide linker.

49. The method according to claim 47, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of an IgG antibody.

50. The glutamine residue to which the peptide linker is conjugated is the C of the IgG antibody. Ｈ The method according to claim 48, wherein the glutamine residue Q295 (EU numbering) is a two-domain glutamine residue.

51. The antibody is a glycosylated IgG antibody, and the IgG antibody is the C Ｈ The method according to claim 47, wherein the residue N297 (EU numbering) of the two domains is glycosylated.

52. The aforementioned antibodies are trastuzumab, brentuximab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, verantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, roncustuximab, mekubotamab, Selected from the group consisting of adecatumumab, D93, gatipotuzumab, rabetuzumab, tusamitamab, rifastuzumab, milbetuximab, sofituzumab, anetuzumab, tisotuzumab, cofituzumab, praluzatamab, radriatuzumab, verantamab, patrizumab, cetuximab, nimotuzumab, matsuzumab, portuzumab, sitatuzumab, tucotsuzumab, and endrecolomab; and / or The method according to claim 47, wherein the antibody specifically binds to an antigen selected from the group consisting of CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM5, NaPi2b, FRα, MUC16, mesoserine, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1.

53. The method according to claim 47, wherein the peptide linker has a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% and is suitable for conjugation to a glycosylated antibody.

54. The method according to claim 47, wherein the transglutaminase is a microbial transglutaminase (MTG) derived from a species of the genus Streptomyces.

55. Formula A-L (wherein A is an antibody or antibody fragment, L is a linker, and the linker is The first payload is a cell membrane-permeable camptothecin cytotoxic molecule; and The second payload includes a non-cell membrane-permeable camptothecin cytotoxic molecule; The cytotoxic molecules in the first and second payloads are exatecan; The second payload has a glycine residue linked to the camptothecin cytotoxic molecule of the second payload; The ADC consists of two first payloads and two second payloads (drug-to-antibody ratio "DAR4"), and The antibody-drug conjugate (ADC) having the aforementioned antibody (which is an IgG antibody).

56. The ADC according to claim 55, wherein the antibody is trastuzumab.

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