Improved cd30 targeting antibody drug conjugates and uses thereof

Abstract

This invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxicin, wherein the belentoxicin comprises a microtubule tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, heavy chain, or all of the heavy chain and the light chain; and (b) at least one pharmaceutical moiety, wherein the pharmaceutical moiety is conjugated to each of the non-natural amino acids via a linker. The invention further relates to a method of manufacturing the same, pharmaceutical compositions comprising the same, and uses thereof.

Description

[Technical Field]

[0001] Cross-Reference to Related Applications. This application claims the benefit of priority to European Patent Application No. 20216838.1, filed on December 23, 2020, the contents of which are incorporated herein by reference in their entirety for all purposes. The present invention relates to antibody-drug conjugates (ADCs) comprising brentuximab and at least one pharmaceutical portion, methods for manufacturing the same, pharmaceutical compositions comprising the same, and uses thereof. [Previous Technology]

[0002] Lymphomas account for approximately 4 to 5% of all cancers and originate from the lymphatic system. In recent years, CD30 has been demonstrated as an important tumor biomarker for targeted therapy in traditional Hodgkin's lymphoma and several non-Hodgkin's lymphomas, such as degenerative large cell lymphoma and peripheral T-cell lymphoma. With the innovative approval of belentuxividotine in 2011, the first CD30-specific biologic drug entered the market and has since become an indispensable treatment option with good overall response. Belentuxividotine consists of the tumor-targeting chimeric IgG1 antibody component belentuxividotine and a linker-load component containing a cathepsin B-cleavable valine-citrulline linker chemically linked to the potent load fraction monomethyl-arestatin E, which effectively induces apoptosis during intracellular delivery and release.

[0003] Belentuxevidorine is an antibody-drug conjugate (ADC), a relatively novel treatment approach that has attracted considerable attention in recent years. These biopharmaceuticals enable the direct delivery of potent cytotoxic drugs to tumor cells, and therefore have the potential to expand the therapeutic window compared to traditional chemotherapy. To date, nine randomly conjugated ADCs have been approved. These molecules are based on conjugation with active carboxylic acids (e.g., Mylotarg, Besponsa, Kadcyla), which randomly react with exposed lysine residues. Alternatively, maleic anhydride has become the most important conjugating agent for conjugating the linker-loaded portion with free cysteine ​​after intrachain disulfide reduction (e.g., belentuxevidorine (available at Adcetris®), Polivy, Padcev, Enhertu, Trodelvy, Blenrep).

[0004] However, belentoxidocin is known to cause sensitive and persistent toxicity as a side effect, such as thrombocytopenia, in clinical practice.

[0005] Therefore, there is still a need for improved ADCs containing belentoxicin, preferably with fewer side effects. The present invention aims to address this need. [Summary of the Invention]

[0006] This requirement is addressed by means of the subject matter as defined in the claims and the specific embodiments described herein.

[0007] Therefore, the present invention relates to antibody-drug conjugates (ADCs) comprising: (a) brentuximab, wherein brentuximab contains a microtubule tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, heavy chain, or all of the heavy chain and the light chain of brentuximab; and (b) at least one pharmaceutical moiety; wherein the pharmaceutical moiety is conjugated to each of the non-natural amino acids via a linker.

[0008] The heavy chain of belentoxicin may have an amino acid sequence comprising SEQ ID NO: 1 or having at least 95% sequence identity with SEQ ID NO: 1, and / or the light chain of belentoxicin may have an amino acid sequence comprising SEQ ID NO: 2 or having at least 95% sequence identity with SEQ ID NO: 2; preferably, belentoxicin is composed of a heavy chain and a light chain, the heavy chain being composed of the amino acid sequence of SEQ ID NO: 1, and the light chain being composed of the amino acid sequence of SEQ ID NO: 2.

[0009] The pharmaceutical portion may be selected from the group consisting of: camptothecin, maytansinoid, calicheamycin, duocarmycin, tubulolysin, amatoxin, dolastatin, and auristatin, such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimer, indolinobenzodiazepine dimer, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTAC, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogues or prodrugs thereof; preferably, the pharmaceutical portion is MMAE.

[0010] The recognition sequence of the tubulin tyrosine ligase may have at least the amino acid sequence X1X2X3X4 (SEQ ID NO: 3), wherein X1 and X2 are any amino acids, X3 is E, D or C, and X4 is E; preferably X2 is G, S, A, V or F, and / or X1 is E, D, A, K or P.

[0011] The identification sequence may be EGEE (SEQ ID No. 4), and more preferably the identification sequence is VDSVEGEGEEEGEE (SEQ ID No. 5), SVEGEGEEEGEE (SEQ ID No. 6), SADGEDEGEE (SEQ ID No. 7), SVEAEAEEGEE (SEQ ID No. 8), SYEDEDEGEE (SEQ ID No. 9), or SFEEENEGEE (SEQ ID No. 10).

[0012] The non-natural amino acid may be a 2-substituted, 3-substituted, or 4-substituted tyrosine, or a tyrosine derivative substituted at the benzyl position. The 3-substituted or 4-substituted tyrosine derivative may be 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-methoxytyrosine, 3-acetyrosine, or 4-aminophenylalanine; preferably, the non-natural amino acid is 3-methoxytyrosine.

[0013] The linker may be cleavable, preferably protease-cleavable, and more preferably cathepsin-cleavable (such as cathepsin B) linker may contain a valine-citrulline moiety. The linker may contain a hydroxylamine group and the non-natural amino acid contains a formyl group located ortho to a hydroxyl group in an aromatic ring (such as 3-methoxytyrosine), wherein the hydroxylamine group of the conjugated linker forms an oxime with the formyl group of the non-natural amino acid.

[0014] Belentoxicin may be conjugated with two, four, six or eight drug fractions, preferably with two or four drug fractions, and more preferably with two drug fractions.

[0015] The linker may have a structure as shown in Structure 1 before being conjugated with a non-natural amino acid: wherein R is one or more pharmaceutical moieties that are optionally conjugated with the hydroxylamine of Structure 1 via one or more cleavage sites, preferably wherein the hydroxylamine of Structure 1 is conjugated with the non-natural amino acid.

[0016] The linker may have a structure as shown in structure 2 or 3 before conjugation with the non-natural amino acid: wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; wherein D is one or more pharmaceutical moieties; and wherein Y is a cleavage site, such as a cleavage site of a cathepsin (such as cathepsin B); preferably wherein the hydroxylamine of structure 2 or 3 is conjugated with the non-natural amino acid.

[0017] The linker may have a structure as shown in structure 4 or 5 before being conjugated with a non-natural amino acid, wherein D is the drug moiety, preferably MMAE:.

[0018] The non-natural amino acid may be 3-methoxytyrosine, and the hydroxylamine group of the linker forms an oxime with the 3-methoxy group of the non-natural amino acid.

[0019] The present invention further relates to a method of manufacturing an ADC as defined herein, comprising: (a) introducing or adding a microtubule tyrosine ligase recognition sequence to the C-terminus of a light chain, a heavy chain, or both a light chain and a heavy chain of belentoxicin; (b) contacting the belentoxicin obtained in step (a) in the presence of microtubule tyrosine ligase and a non-natural amino acid, under conditions suitable for microtubule tyrosine ligase to link belentoxicin to the non-natural amino acid; and (c) conjugating an optional cleavable linker comprising a pharmaceutical moiety to the linked belentoxicin obtained in step (b).

[0020] The present invention further relates to an ADC that can be obtained by a method of manufacturing an ADC as defined herein. The present invention further relates to an ADC that can be obtained by a method of manufacturing an ADC as defined herein.

[0021] The present invention further relates to a pharmaceutical composition comprising the ADC of the present invention.

[0022] This invention further relates to the use of the ADC of the present invention or the pharmaceutical composition of the present invention in a method of treating a disease. Preferably, the disease is associated with overexpression of CD30. More preferably, the disease is selected from the group consisting of: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell cancer; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL).

Implementation Method

[0045] The present invention will be described in detail below and further illustrated by the accompanying examples and figures.

[0046] Lymphomas account for approximately 4 to 5% of all cancers and originate from the lymphatic system. In recent years, CD30 has been demonstrated as an important tumor biomarker for targeted therapy in traditional Hodgkin's lymphoma and several non-Hodgkin's lymphomas, such as degenerative large cell lymphoma and peripheral T-cell lymphoma. With the innovative approval of belentuximab vedotin (Adcetris®) in 2011, the first CD30-specific biologic drug entered the market and has since become an indispensable treatment option with good overall response. Belentuximab vedotin consists of belentuximab, a chimeric IgG1 antibody component targeting tumors, and a linker-load component containing a cathepsin B-cleavable valine-citrulline linker chemically linked to the potent load fraction monomethyl-arestatin E, which effectively induces apoptosis during intracellular delivery and release.

[0047] However, belentoxidocin is known to cause sensitive and persistent toxicity as a side effect, such as thrombocytopenia, in clinical practice.

[0048] The inventors explain that the use of tubulin tyrosine ligase surprisingly enables highly specific and stoichiometrically defined conjugation of the drug moiety with belentuximab, which has been modified by adding a TTL recognition sequence to the C-terminus of the light chain and / or heavy chain. The TTL can be used to conjugate non-natural amino acids (such as 3-methoxytyrosine) with the TTL recognition sequence. The drug moiety (e.g., MMAE) can be specifically conjugated with non-natural amino acids via linkers (e.g., linkers containing active groups that can react with active groups on non-natural amino acids). For example, the linker may contain hydroxylamine, which reacts with the methoxy group of the non-natural amino acid to form an oxime (see Example 1). In summary, the inventors demonstrate that the combination of the functional components belentuximab and MMAE of belentuximab vitolamine using the conjugation strategy described herein surprisingly enhances the efficacy of belentuximab vitolamine. The ADC of the present invention surprisingly exhibits higher in vitro stability (see Example 2), higher in vivo efficacy (see Example 4), higher in vivo stability (see Example 5), and improved toxicological properties (see Example 6). Therefore, the side effects of belentoxidocin are remarkably reduced.

[0049] Therefore, the present invention relates to antibody-drug conjugates (ADCs) comprising: (a) brentuximab, wherein brentuximab contains a microtubule tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, heavy chain, or all of the heavy chain and the light chain of brentuximab; and (b) at least one pharmaceutical moiety; wherein the pharmaceutical moiety is conjugated to each of the non-natural amino acids via a linker.

[0050] As used herein, the term "antibody-drug conjugate" (ADC) generally refers to the linking of an antibody (such as belentoxicin or its antigen-binding fragment) to another reagent (such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc.). The linking can be covalent or non-covalent interaction such as by electrostatic force, preferably covalent. Various linkers known in the art and described herein can be used to form antibody-drug conjugates. The ADC of the present invention can also be described by having the formula Ab-(L-(D)x)y. "Ab" represents the antibody (such as belentoxicin or its antigen-binding fragment), "L" represents the linker, and "D" represents the drug moiety. "x" can be an integer from 1 to 10. "y" can be an integer from 1 to 10. Therefore, x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Therefore, y can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. As a purely illustrative example, x or y can be an integer from 1 to 8, 1 to 6, 1 to 4, or 1 to 2. "Ab": ​​Antibody belentoxigenics

[0051] As outlined herein, the ADC of the present invention can be described by having the formula Ab-(L-(D)x)y. The "Ab portion" of the ADC of the present invention is belenutoxine. Belenutoxine, also known as cAC10 or SGN-30, is an antibody well known to those skilled in the art (see, for example, Wahl et al. (2002), Cancer Res, 62:3736-3742). Belenutoxine is a monoclonal antibody that specifically binds to CD30 on the surface of target cells. Binding to CD30 initiates the internalization of belenutoxine, which then enters the lysosomal compartment. Intracellularly, the drug portion of the ADC can be released from the monoclonal antibody, for example, via cleavage or degradation of the linker. The ADC of the present invention contains belenutoxine.

[0052] CD30, also known as TNFRSF8, is a cell membrane protein belonging to the tumor necrosis factor receptor family and a tumor marker. This receptor is expressed by activated T cells and B cells, but not by quiescent T cells and B cells. TRAF2 and TRAF5 can interact with this receptor and mediate the signaling pathway leading to NF-κB activation. It is a positive regulator of apoptosis and has also been shown to limit the proliferative potential of self-reactive CD8 effector T cells and protect the body from autoimmune influences. Two alternatively spliced ​​transcriptomorphs of this gene with different isoforms have been reported. CD30 is associated with degenerative large cell lymphoma. It is expressed in embryonal carcinomas but not in seminomas, and is therefore a useful marker for differentiating these germ cell tumors. CD30 and CD15 are also expressed in Reed-Sternberg cells, a typical Hodgkin's lymphoma.

[0053] Each heavy chain of belentoxix, as an approved drug and as used herein, has the amino acid sequence of SEQ ID NO: 1 (also shown in Figure 21). Antibodies whose heavy chains have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1 may also be used herein. For example, as shown in Figure 21, the last residue K at position 447 may be omitted if necessary. Such a heavy chain can be considered a variant of the belentoxix heavy chain. Similarly, the first two or three N-terminal residues of, for example, the belentoxix heavy chain may be deleted. Compared to belentoxix, such a heavy chain would have three or four mutations and therefore have a sequence identity of approximately 99.2% (four amino acid differences) to 99.8% (one amino acid difference) with the belentoxix heavy chain.

[0054] Each light chain of belentoxix, as an approved drug and as used herein, has the amino acid sequence of SEQ ID NO: 2 (also shown in Figure 21). Antibodies whose light chains have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2 may also be used herein. For example, the last residue C at position 218 may be omitted if desired. Such a light chain can be considered a variant of the belentoxix light chain. Similarly, the first two or three N-terminal residues of, for example, the belentoxix light chain may be deleted. Compared to belentoxix, such a light chain would have three or four mutations and thus a sequence identity of 98.2% (four amino acid differences) to 99.5% (one amino acid difference) with the belentoxix light chain.

[0055] In one particular embodiment, belentoxicin comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 and a light chain containing the amino acid sequence of SEQ ID NO: 2. In a further particular embodiment, belentoxicin comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and the light chain comprising the amino acid sequence of SEQ ID NO: 2.

[0056] Generally, when used herein, in the context of two or more nucleic acid or polypeptide sequences, terms such as "percentage (%) identical" or "percentage (%) consistent" refer to the degree to which two or more sequences or subsequences are identical. Two sequences are "identical" if they have the same amino acid or nucleotide sequence in the region being compared. When comparing and aligning maximum correspondence within a comparison range or specified region, two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identical in the specified region, or, when not specified, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical across the entire sequence), as measured by manual alignment and visual inspection, using one of the following sequence comparison algorithms. Optionally, consistency is present in regions of at least about 30 nucleotides (or 10 amino acids) in length, or more preferably in regions of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0057] The percentage of sequence homology or sequence identity can be determined, for example, using the BLASTP program (blastp version 2.2.5, November 16, 2002) (Altschul et al., Nucleic Acids Res, 1997). In this specific embodiment, the homology percentage is based on the alignment of the entire polypeptide sequence, including the original peptide sequence (matrix: BLOSUM 62; gap deduction: 11.1; cutoff value set at 10⁻³), preferably using a wild-type protein scaffold as a reference in pairwise comparisons. It is calculated by dividing the number of "positive" (homological amino acids) displayed as results in the BLASTP program output by the percentage of the total number of amino acids selected by the program for alignment. TTL recognition sequences and conjugation of non-natural amino acids with belentoxicin are also considered.

[0058] In the ADC of the present invention, belentoxicin may be fused with one or more TTL recognition sequences, for example, at the C-terminus of the light chain, heavy chain, or both light and heavy chains. This TTL recognition sequence allows the TTL to conjugate the non-natural amino acid to the TTL recognition sequence at the C-terminus of the light chain and / or heavy chain of belentoxicin. Therefore, the belentoxicin included in the ADC of the present invention contains a microtubule tyrosine ligase recognition sequence ("TTL recognition sequence") at the C-terminus of the light chain, heavy chain, or all heavy and light chains of belentoxicin. In the ADC of the present invention, the non-natural amino acid is linked to this TTL recognition sequence. The drug moiety is conjugated to this non-natural amino acid via a linker. When used herein, "Tub-tag" refers to a TTL recognition sequence or a TTL recognition sequence, preferably SEQ ID NO: 4 or SEQ ID NO: 5.

[0059] As used herein and throughout the specification, the term "microtubule-tyrosine ligase," sometimes abbreviated as "TTL," encompasses a polypeptide capable of functionalizing a polypeptide, that is, covalently linking a non-natural amino acid, as defined herein, to the polypeptide. For this function, the polypeptide preferably contains a TTL recognition sequence. This term includes TTLs from eukaryotes, preferably mammals, and more preferably from gray wolves. A preferred TTL is shown in SEQ ID NO: 13. This term also includes TTLs having 70%, 80%, 90%, or 95% or more amino acid sequence identity with the TTL shown in SEQ ID NO: 13 throughout its entire amino acid sequence. Preferably, such polypeptides having the aforementioned shared amino acid sequence identity have TTL activity. TTL activity can be tested as known in the art or as described herein. The percentage of sequence identity can be determined, for example, as described herein. Preferably, the amino acid sequence shown in SEQ ID NO: 13 is used as a reference in pairwise comparisons.

[0060] As used herein and throughout this specification, the term “functionalization” in all grammatical forms used herein refers to “covalently linking a non-natural amino acid” to a polypeptide (such as belentoxigenic amino acid). Without wishing to be bound by any particular theory, it may be conceivable that TTL adds a non-natural amino acid, as defined herein, to the final C-terminal amino acid of the TTL recognition sequence.

[0061] Methods for modifying proteins using tubulin-tyrosine ligase (TTL) are described in WO 2016 / 066749 and WO 2017 / 186855, which are incorporated herein by reference. For a TTL to link a non-natural amino acid to an antibody of interest (such as an ADC described herein), the TTL requires a recognition sequence. The ADC described herein may be modified to include a recognition sequence for tubulin-tyrosine ligase (TTL) at the C-terminus of the light chain, heavy chain, or both the light and heavy chains of belentoxicin, which comprises at least the amino acid sequence X4X3X2X1. The terms "recognition sequence" or "recognition motif" are used interchangeably herein and refer to an amino acid segment recognized by the TTL. Such recognition sequences are known in the art; see, for example, Ruediger et al. (1994), Eur. J. Biochem. 220, 309-320 or Prota et al. (2013), J. Cell. Biol. 200, No. 3, 259-270. Furthermore, those skilled in the art can readily test whether an amino acid sequence of interest is a TTL-recognized sequence by applying, for example, the "peptide tyrosineing by TTL" test described by Ruediger et al. Being "recognized" by a TTL includes the binding of the TTL to the recognition motif. The recognition motif advantageously comprises at least four amino acids, which are designated herein as X4, X3, X2, and X1. Generally, unless otherwise stated herein, "X" may represent any amino acid. Amino acids include, but are not limited to, twenty "standard" amino acids: isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W), valine (Val, V), alanine (Ala, A), aspartic acid (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), serine (Ser, S), tyrosine (Tyr, Y), arginine (Arg, R), and histidine (His, H). This invention also includes, but is not limited to, D-type amino acids, β-amino acids, amino acids with side chains, and all non-natural amino acids known to those skilled in the art.

[0062] The recognition sequence of the tubulin tyrosine ligase may have at least the amino acid sequence X1X2X3X4 (SEQ ID NO: 3), wherein X1 and X2 are any amino acids, X3 is E, D or C, and X4 is E; preferably, X2 is G, S, A, V or F, and / or X1 is E, D, A, K or P. The recognition sequence may be EGEE (SEQ ID No. 4), and preferably VDSVEGEGEEEGEE (SEQ ID No. 5), SVEGEGEEEGEE (SEQ ID No. 6), SADGEDEGEE (SEQ ID No. 7), SVEAEAEEGEE (SEQ ID No. 8), SYEDEDEGEE (SEQ ID No. 9), or SFEEENEGEE (SEQ ID No. 10). In one specific embodiment, the recognition sequence is VDSVEGEGEEEGEE (SEQ ID No. 5).

[0063] The number of TTL recognition sequences present in belentoxicin contained in the ADC of the present invention determines the drug-to-antibody ratio (DAR). Although the drug-to-antibody ratio (DAR) has a defined stoichiometric value for a particular conjugate molecule (e.g., y multiplied by x in the formula Ab-(L-(D)x)y), it should be understood that when used to describe samples containing many molecules, this value may be an average value due to some degree of heterogeneity (typically related to the conjugation step). Therefore, in the context of any of the antibody-drug conjugates described herein, the average loading of a sample of antibody-drug conjugates is referred to as the drug-to-antibody ratio, or "DAR". However, preferably, the present invention provides ADCs in which substantially all or all of the ADCs have the desired DAR, or in other words, a stoichiometric value.

[0064] The ADC of the present invention is based on belentoxicin, a monoclonal IgG antibody. The IgG antibody comprises two "light chains" and two "heavy chains," or is composed of them. Since TTL recognition sequences can only be fused to the C-terminus of the light chain, heavy chain, or both the light and heavy chains, the maximum number of TTL recognition sequences in belentoxicin is limited to four. When either the heavy chain or light chain contains a TTL recognition sequence at its C-terminus, the belentoxicin contained in the ADC of the present invention has two TTL recognition sequences. When both the heavy chain and light chain contain TTL recognition sequences at their C-termini, the belentoxicin contained in the ADC of the present invention has four TTL recognition sequences.

[0065] In the ADC of the present invention, the TTL recognition sequence may be directly fused to the C-terminus of the antibody belentoxixine chain. See here, belentoxixine heavy chain (SEQ ID NO: 12): wherein the TTL recognition sequence is directly fused to the C-terminus of the heavy chain (i.e., no amino acid residues are present therein). Alternatively, a (peptide) linker may be arranged between the TTL recognition sequence and the antibody chain. Therefore, the belentoxixine light chain, heavy chain, or both may include an amino acid linker sequence arranged between the TTL recognition sequence and the corresponding C-terminus. As an illustrative example, see the light chain of SEQ ID NO: 11, wherein the TTL recognition sequence is fused to the C-terminus of the belentoxixine light chain via a GGGGS (G4S) linker. The amino acid linker may have any suitable length, as long as the function of the TTL recognition sequence is preserved. For example, amino acid linkers may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues. See again the light chain of SEQ ID NO: 11, where the GGGGS (G4S) linker is positioned between the TTL recognition sequence and the C-terminus of belentoxigenic. Other standard linkers, such as (G4S)2 or (G4S)3, may also be used, which are commonly used as linkers for fusing recombinant antibody molecules of different chains (such as single-chain Fv fragments) together. Thus, in these examples, the linker length may be 10 amino acid residues ((G4S)2) or 15 amino acid residues ((G4S)3). However, the linker is by no means limited to a maximum length of 15 amino acid residues, but as disclosed above, the amino acid linker sequence may contain even more than 15 amino acids.

[0066] Also according to the above disclosure, the ADC of the present invention can be constructed such that no antibody chain contains a peptide linker between the TTL recognition sequence and the corresponding antibody chain. In such an ADC, the TTL recognition sequence can be directly fused to both the light and heavy chains of belentoxicin. However, the ADC of the present invention can also be constructed such that one of the antibody chains contains a peptide linker between the TTL recognition sequence and the corresponding antibody chain. In such an ADC, the TTL recognition sequence can be fused to each light chain via a linker (e.g., the GGGGS-linker in the light chain of SEQ ID NO: 11), and the TTL recognition sequence can be directly fused to each of the two heavy chains of belentoxicin. Finally, the ADC of the present invention can also be constructed such that both the antibody light chain and the antibody heavy chain contain a peptide linker between the TTL recognition sequence and the corresponding antibody chain. In such an ADC, the TTL recognition sequence can be fused to each of the two light chains and each of the two heavy chains of belentoxicin via a linker (e.g., the GGGGS-linker or the (G4S)2 linker).

[0067] In one specific embodiment, belentoxicin includes two TTL recognition sequences at the C-terminus of the light chain. The TTL recognition sequences can be fused to the C-terminus of the light chain via an amino acid linker sequence (such as GGGGS). The TTL recognition sequences can bind directly to the C-terminus of the light chain. The belentoxicin heavy chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 1, and the belentoxicin light chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 11. The belentoxicin heavy chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 1, and the belentoxicin light chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 14. The belentoxicin heavy chain may be composed of polypeptides having the sequence shown in SEQ ID NO: 1, and the belentoxicin light chain may be composed of polypeptides having the sequence shown in SEQ ID NO: 11. The belentoxigenic heavy chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 1, and the belentoxigenic light chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 14.

[0068] In one specific embodiment, belentoxicin includes four TTL recognition sequences at the C-terminus of both the light and heavy chains. The TTL recognition sequences can be fused to the C-terminus of the light chain via an amino acid linker sequence (such as GGGGS), and can also bind directly to the C-terminus of the heavy chain. The heavy chain of belentoxicin may comprise a polypeptide having the sequence shown in SEQ ID NO: 12, and the light chain of belentoxicin may comprise a polypeptide having the sequence shown in SEQ ID NO: 11. The belentoxicin heavy chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 12, and the belentoxicin light chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 14. The belentoxicin heavy chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 12, and the belentoxicin light chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 11. The belentoxicin heavy chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 12, and the belentoxicin light chain may be composed of a polypeptide having the sequence shown in SEQ ID NO: 14.

[0069] In one specific embodiment, belentoxicin includes two TTL recognition sequences at the C-terminus of the heavy chain. The TTL recognition sequences can bind directly to the C-terminus of the heavy chain. Alternatively, the TTL recognition sequences can bind to the C-terminus of the heavy chain via an amino acid linker sequence (such as GGGGS). The belentoxicin heavy chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 12, and the belentoxicin light chain may comprise a polypeptide having the sequence shown in SEQ ID NO: 2. The belentoxicin heavy chain may be composed of polypeptides having the sequence shown in SEQ ID NO: 12, and the belentoxicin light chain may be composed of polypeptides having the sequence shown in SEQ ID NO: 2.

[0070] However, the number of TTL recognition sequences is not necessarily equal to the number of drug moieties conjugated with non-natural amino acids, i.e., not necessarily equal to DAR. The conjugation (via linkers) of more than one drug moieties with non-natural amino acids is also covered in this invention. For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably two drug moieties conjugated with one non-natural amino acid via a linker. In this case, the linker can act as a scaffold, and 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably two drug moieties conjugated with this scaffold. The linker is thus conjugated with the non-natural amino acid. An exemplary linker as a scaffold for two drug moieties is structure 3 or 5. Therefore, the antibody can be conjugated with two, four, six, or eight, preferably two or four drug moieties, more preferably two drug moieties. Non-natural amino acids

[0071] The non-natural amino acid is the anchor point of the linker in the "Ab" portion of the ADC of the present invention. In this regard, the "ADC chemical formula" described herein is reiterated: Ab-(L-(D)x)y. Therefore, the non-natural amino acid conjugated at the C-terminus of a polypeptide (such as belentoxicin contained in the ADC of the present invention) with a TTL recognition sequence at its C-terminus can contain a functional group that can react with the functional group of the linker L to form a covalent bond.

[0072] Generally, non-natural amino acids can be considered as tyrosine derivatives. Preferably, tyrosine derivatives contain active groups that enable the drug to be conjugated via linkers.

[0073] Tyrosine derivatives may (further) contain non-natural functional groups, which are preferably used for chemoselective or bioorthogonal modification. These functional groups may be suitable for click chemistry. The term "click chemistry" refers to the chemical principles introduced by Kolb, Finn, and Sharpless in 2001 and encompasses a powerful set of linking reactions that can rapidly and reliably generate covalent bonds by linking small units containing reactive groups together. Click chemistry reactions are typically modular, wide-ranging, produce high chemical yields, generate harmless byproducts, are stereospecific, and / or can be carried out using readily available starting materials and reagents under simple physiological reaction conditions. Furthermore, click chemistry reactions preferably do not use toxic solvents or use affinity or easily removable solvents (preferably water), and / or provide simple product separation by non-chromatographic methods (crystallization or distillation). The pronounced exothermic nature of the reaction makes the reactants "spring-loaded."

[0074] Click chemistry reactions include, for example, the following: cycloaddition reactions, particularly those from the 1,3-dipolar family, hetero-Diels-Alder reactions; nucleophilic ring-opening reactions, such as those involving strained heterocyclic electrophiles, such as epoxides, aziridines, cyclic sulfates, cyclic aminosulfonates, aziridine-onium ions, and epithionium ions; non-aldecorative carbonyl chemistry (e.g., the formation of oxime ethers, hydrazones, and aromatic heterocycles); and additions to carbon-carbon multiple bonds; for example, oxidation reactions, such as epoxidation, dihydroxylation, aziridineation, and additions to nitroso and hyposulfonyl halides, as well as certain Michael addition reactions. The general principles of click chemistry reactions have been described by Kolb, Finn, and Sharpless (2001). The selection of a click chemistry suitable for linking a desired drug portion to a tyrosine derivative or non-natural amino acid via a linker is known to those skilled in the art, which covalently binds to belentoxicin contained in the ADC of the present invention.

[0075] As used herein, the term "click chemistry handle" refers to a reactant or reactive group that can participate in a click chemistry reaction. Such a reactant or reactive group is preferably a non-natural (unnatural) functional group for chemoselective or bioorthogonal modification; however, it can also be a natural functional group for chemoselective or bioorthogonal modification. For example, strained alkynes, such as cyclooctyne, are click chemistry handles because they can participate in strain-enhanced cycloaddition reactions, such as strain-enhanced azide-alkyne cycloaddition reactions (SPAAC). Generally, click chemistry reactions require at least two molecules containing click chemistry handles that can react with each other. Such a pair of click chemistry handles that react with each other are sometimes referred to herein as "partner click chemistry handles." For example, an azide is a partner click chemistry handle for cyclooctyne or any other alkyne. In the context of this invention, the click chemical handle is preferably selected from the group consisting of: terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyl groups, β-arylethylamine, phosphine, acehydrazine, hydrazine, thiols, tetrahydrazine, alkenes, cyclooctene, norethene, tetrahydrazine, nitrones, cyanobenzothiazoles, and cyclooctyne. Other suitable click chemical handles are readily available to those skilled in the art.

[0076] In the case of click chemical conjugation, conjugation is a covalent bond formed via a reaction of the click chemical handle. In some specific embodiments, the association is covalent, and the entities are referred to as "conjugated" to each other.

[0077] It should be noted that the present invention is not limited to the exemplary click chemical handles described above, and additional click chemical handles, reactive click chemical handle pairs, and reaction conditions for such click chemical handle pairs will be apparent to those skilled in the art.

[0078] Other suitable methods for conjugating the linker with the non-natural amino acid contained in belentoxicin contained in the ADC of the present invention include the following: Staudinger reaction (e.g., Staudinger-linking, Staudinger-phosphite reaction, Staudinger-phosphite reaction), strain-promoted cycloaddition reaction, tetrahydrothiazole linking, electron-demanding Diels-Adel reaction, tetrahydrothiazole formation reaction of aldehyde or ketone with 1,2-aminothiol, acezoline formation reaction of aldehyde, oxime formation, hydrazone formation, The reactions can include acetal formation of ketones with 1,2-amino alcohols, aldehydes, or ketones with 1,2-diols; Pictet-Spengler reactions; captured Knoevenagel linkages; tandem Knoevenagel condensations; addition reactions of thiols with alkenes or alkynes; cross metathesis; metal catalysis, especially Pd-, Cu, Ni, and Fe-catalyzed cross-conjugation with electron-withdrawing tyrosine derivatives, preferably oxime formation.

[0079] It is conceivable that the drug moiety may be linked to a non-natural amino acid via a linker covalently bonded to belentoxicin, for example by click chemistry or any other suitable method described herein. Thus, the drug moiety may be conjugated to a non-natural amino acid via a linker via a non-peptide bond; however, alternatively, it may also be conjugated to a non-natural amino acid via a linker via a peptide bond.

[0080] The non-natural amino acid may be a 2-substituted, 3-substituted, or 4-substituted tyrosine, or a tyrosine derivative substituted at the benzyl position. The 3-substituted or 4-substituted tyrosine derivative may be 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-methoxytyrosine, 3-acetyrosine, or 4-aminophenylalanine. The 3-substituted tyrosine may be 3-nitrotyrosine. The 3-substituted tyrosine may be 3-aminotyrosine. The 3-substituted tyrosine may be 3-azidotyrosine. The 3-substituted tyrosine may be 3-methoxytyrosine. The 3-substituted tyrosine may be 3-acetyrosine. The substituted tyrosine may be substituted phenylalanine. The 4-substituted tyrosine may be 4-aminophenylalanine. A particularly good non-natural amino acid is 3-methoxytyrosine.

[0081] As used herein and throughout the specification, the terms "optionally substituted" or "substituted" mean that one or more (such as 1 to a maximum number of hydrogen atoms bonded to the group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms may be replaced by groups other than hydrogen, such as alkyl (preferably C1-6 alkyl), alkenyl (preferably C2-6 alkenyl), ynyl (preferably C2-6 ynyl), aryl (preferably 3 to 14 aryl), heteroaryl (preferably 3 to 14 heteroaryl), cycloalkyl (preferably 3 to 14 cycloalkyl), heterocyclic (preferably 3 to 14 heterocyclic) base), halogen, CN, azido, NO2, OR71, N(R72)(R73), ON(R72)(R73), N+(O-)(R72)(R73), S(O)02R71, S(O)02OR71, OS(O)02R71, OS(O)02OR71, S(O )02N(R72)(R73), OS(O)02N(R72)(R73), N(R71)S(O)02R71, NR71S(O)02OR71, NR71S(O)02N(R72)(R73), C(=W1)R71, C(=W1)W1R71, W1C (=W1)R71, and -W1C(=W1)W1R71; wherein R71, R72, and R73 are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, 3 to 7-membered cycloalkyl, 5 or 6-membered aryl, 5 or 6-membered heteroaryl, and 3 to 7-membered heterocyclic, wherein the alkyl, alkenyl, ynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups are each optionally substituted by one, two, or three substituents selected from the group consisting of: C1-3 alkyl, halogen, CF3, CN, azide, NO2, OH, O (C1-3 alkyl), S (C1-3 alkyl), NH2, NH (C1-3 alkyl), N ( C1-3 alkyl)2, NHS(O)2(C1-3 alkyl), S(O)2NH2-z(C1-3 alkyl)z, C(=O)OH, C(=O)O(C1-3 alkyl), C(=O)NH2-z(C1-3 alkyl)z, NHC(=O)(C1-3 alkyl), NHC(=NH)NHz-2(C1-3 alkyl)z, and N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein z is 0, 1, or 2, and the C1-3 alkyl is methyl, ethyl, propyl, or isopropyl; W1 is independently selected from O, S, and NR84, wherein R84 is -H or C1-3 alkyl.

[0082] As used herein and throughout the specification, the term "alkyl" refers to a single radical of a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. In some specific embodiments, the alkyl group used in the present invention comprises 1 to 20 carbon atoms (C1-20 alkyl). In another specific embodiment, the alkyl group used comprises 1 to 15 carbon atoms (C1-15 alkyl). In another specific embodiment, the alkyl group used comprises 1 to 10 carbon atoms (C1-10 alkyl). In another specific embodiment, the alkyl group used comprises 1 to 8 carbon atoms (C1-8 alkyl). In another specific embodiment, the alkyl group used comprises 1 to 6 carbon atoms (C1-6 alkyl). In another specific embodiment, the alkyl group used contains 1 to 5 carbon atoms (C1-5 alkyl). In another specific embodiment, the alkyl group used contains 1 to 4 carbon atoms (C1-4 alkyl). In another specific embodiment, the alkyl group used contains 1 to 3 carbon atoms (C1-3 alkyl). In another specific embodiment, the alkyl group used contains 1 to 2 carbon atoms (C1-2 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tributyl, n-pentyl, isopentyl, dipentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, dihexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc., which may have one or more substituents. Alkyl substituents include, but are not limited to, any substituents described herein, which result in the formation of a stable moiety. In some specific embodiments, the alkyl chain is straight-chain. In some specific embodiments, the alkyl chain is branched. In some embodiments, the alkyl chain is substituted. In some embodiments, the alkyl chain is unsubstituted. In some embodiments, the alkyl chain is straight-chain and is either substituted or unsubstituted. In some embodiments, the alkyl chain is branched and is either substituted or unsubstituted.

[0083] As used herein and throughout the specification, the term "alkylene" refers to a diradical of a saturated straight-chain or branched hydrocarbon. Preferably, the alkylene contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methylene, ethyl (i.e., 1,1-ethyl, 1,2-ethyl), propyl (i.e., 1,1-propyl, 1,2-propyl (-CH(CH3)CH2-), 2,2-propyl (-C(CH3)2-), and 1,3-propyl), butyl isomers (e.g., 1,1-butyl, 1,2-butyl, 2,2-butyl, 1,3-butyl, 2,3-butyl (cis or trans or mixtures thereof), 1,4-butyl, 1,1 - Isobutyl, 1,2-isobutyl, and 1,3-isobutyl), pentyryl isomers (e.g., 1,1-pentyl, 1,2-pentyl, 1,3-pentyl, 1,4-pentyl, 1,5-pentyl, 1,1-isopentyl, 1,1-dimethylpentyl, 1,1-neopentyl), pentyryl isopentyl (e.g., 1,1-hexyl, 1,2-hexyl, 1,3-hexyl, 1,4-hexyl, 1,5-hexyl, 1,6-hexyl, and 1,1-isohexyl), etc. The pentyryl alkyl group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. Pentyryl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0084] As used herein and throughout the specification, the terms "halogen" or "halogen" refer to fluorine, chlorine, bromine, or iodine groups.

[0085] As used herein and throughout the description, the term "azido" refers to N3.

[0086] As used herein and throughout the specification, the term "alkenyl" refers to a single radical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4 carbon-carbon double bonds. More preferably, the alkenyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, the alkenyl group comprises 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. In some embodiments, the alkenyl group used in this invention comprises 2 to 20 carbon atoms (C2-20 alkenyl). In some embodiments, the alkenyl group used in this invention comprises 2 to 15 carbon atoms (C2-15 alkenyl). In another embodiment, the alkenyl group used comprises 2 to 10 carbon atoms (C2-10 alkenyl). In other embodiments, the alkenyl group comprises 2 to 8 carbon atoms (C2-8 alkenyl). In other embodiments, the alkenyl group comprises 2 to 6 carbon atoms (C2-6 alkenyl). In other embodiments, the alkenyl group comprises 2 to 5 carbon atoms (C2-5 alkenyl). In other specific embodiments, the alkenyl group contains 2 to 4 carbons (C2-4 alkenyl). In other specific embodiments, the alkenyl group contains 2 to 3 carbons (C2-3 alkenyl). In other specific embodiments, the alkenyl group contains 2 carbons (C2 alkenyl). The carbon-carbon double bond may be cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octyl Alkenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, etc. If the alkenyl group is connected to a nitrogen atom, the nitrogen atom cannot be at the α-position of the double bond. In some specific embodiments, the alkenyl chain is straight-chain.In some embodiments, the alkenyl chain is branched. In some embodiments, the alkenyl chain is substituted. In some embodiments, the alkenyl chain is unsubstituted. In some embodiments, the alkenyl chain is linear and is either substituted or unsubstituted. In some embodiments, the alkenyl chain is branched and is either substituted or unsubstituted. Alkenyl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0087] As used herein and throughout the specification, the term "alkenyl" refers to a bis-monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4 carbon-carbon double bonds. More preferably, the alkenyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, the enyl group comprises 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably comprising 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be cis (Z) or trans (E) configurations. Exemplary enyl groups include vinylidene-1,2-diyl, vinylidene, 1-propen-1,2-diyl, 1-propen-1,3-diyl, 1-propen-2,3-diyl, allyl, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. If the enyl group is attached to a nitrogen atom, the nitrogen atom cannot be at the α-position of the double bond. The enyl group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. Enyl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0088] As used herein and throughout the specification, the term "alkynyl" refers to a single radical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is odd, the result of the division is rounded to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 4, i.e., 1, 2, 3 or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, the alkynyl group comprises 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. In some embodiments, the alkynyl group used in this invention comprises 2 to 20 carbon atoms (C2-20 alkynyl). In some embodiments, the alkynyl group used in this invention comprises 2 to 15 carbon atoms (C2-15 alkynyl). In another embodiment, the alkynyl group used comprises 2 to 10 carbon atoms (C2-10 alkynyl). In other embodiments, the alkynyl group comprises 2 to 8 carbon atoms (C2-8 alkynyl). In other embodiments, the alkynyl group comprises 2 to 6 carbon atoms (C2-6 alkynyl). In other specific embodiments, the alkynyl group contains 2 to 5 carbon atoms (C2-5 alkynyl). In other specific embodiments, the alkynyl group contains 2 to 4 carbon atoms (C2-4 alkynyl). In other specific embodiments, the alkynyl group contains 2 to 3 carbon atoms (C2-3 alkynyl). In other specific embodiments, the alkynyl group contains 2 carbon atoms (C2 alkynyl). Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-hepynyl, 2-hepynyl, 3-hepynyl, 4-hepynyl, 5-hepynyl, 6-hepynyl, 1-octyynyl, 2-octyynyl, 3-Octyrynyl, 4-octyrynyl, 5-octyrynyl, 6-octyrynyl, 7-octyrynyl, 1-nonyrynyl, 2-nonyrynyl, 3-nonyrynyl, 4-nonyrynyl, 5-nonyrynyl, 6-nonyrynyl, 7-nonyrynyl, 8-nonyrynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, etc., which may have one or more substituents.The alkynyl substituent includes, but is not limited to, any substituents described herein that result in the formation of a stable moiety. If the alkynyl group is attached to a nitrogen atom, the nitrogen atom cannot be at the α-position of the triple bond. In some embodiments, the alkynyl chain is straight-chain. In some embodiments, the alkynyl chain is branched. In some embodiments, the alkynyl chain is substituted. In some embodiments, the alkynyl chain is unsubstituted. In some embodiments, the alkynyl chain is straight-chain and is either substituted or unsubstituted. In some embodiments, the alkynyl chain is branched and is either substituted or unsubstituted.

[0089] As used herein and throughout the specification, the term "acetylenic" refers to a bis-monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. Generally, the maximum number of carbon-carbon triple bonds in an acetylenic group can be equal to the integer obtained by dividing the number of carbon atoms in the acetylenic group by 2, and if the number of carbon atoms in the acetylenic group is odd, the result of the division is rounded to the next integer. For example, for an acetylenic group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the acetylenic group has 1 to 4, i.e., 1, 2, 3 or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the acetylenic group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, the phenylene group comprises 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary phenylene groups include acetylene-1,2-diyl, 1-propyne-1,3-diyl, 1-propyne-3,3-diyl, 1-butyne-1,3-diyl, 1-butyne-1,4-diyl, 1-butyne-3,4-diyl, 2-butyne-1,4-diyl, etc. If the phenylene group is connected to a nitrogen atom, the nitrogen atom cannot be at the α-position of the triple bond. The ynyl group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. The ynyl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0090] As used herein and throughout the specification, the terms "cycloalkyl," "cycloaliphatic," "carbocyclic," or "carbocyclic" refer to cyclic non-aromatic forms of "alkyl" and "alkenyl" having a preferred number of 3 to 14 carbon atoms, such as 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 7 carbon atoms. In some embodiments, the cycloalkyl used in this invention contains 3 to 14 carbon atoms (C3-14 cycloalkyl). In some embodiments, the cycloalkyl used in this invention contains 3 to 12 carbon atoms (C3-12 cycloalkyl). In another embodiment, the cycloalkyl used in this invention contains 3 to 10 carbon atoms (C3-10 cycloalkyl). In another embodiment, the cycloalkyl used in this invention contains 3 to 8 carbon atoms (C3-8 cycloalkyl). In yet another embodiment, the cycloalkyl used in this invention contains 3 to 7 carbon atoms (C3-7 cycloalkyl). In another specific embodiment, the cycloalkyl group used in this invention contains 3 to 6 carbon atoms (C3-6 cycloalkyl). In another specific embodiment, the cycloalkyl group used in this invention contains 3 to 5 carbon atoms (C3-5 cycloalkyl). In another specific embodiment, the cycloalkyl group used in this invention contains 3 to 4 carbon atoms (C3-4 cycloalkyl). In another specific embodiment, the cycloalkyl group used in this invention contains 3 carbon atoms (C3 cycloalkyl). Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. The term "cycloalkyl" also means including its bicyclic and tricyclic forms. If a bicyclic ring is formed, it is preferred that the rings are connected to each other at two adjacent carbon atoms; however, the two rings may be connected via the same carbon atom, i.e., it forms a spirocyclic system or a "bridged" ring system. Preferred examples of cycloalkyl groups include C3-C8 cycloalkyl groups, particularly cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, and bicyclo[4.2.0]octyl. Cycloalkyl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0091] As used herein and throughout the specification, the term "cyclopropyl" refers to the cyclopropyl group as defined above, in which one hydrogen atom is removed to form a diradical. The cyclopropyl group may consist of two atoms or portions connected via the same carbon atom (1,1-cyclopropyl, i.e., a paired diradical) or via two carbon atoms (1,2-cyclopropyl).

[0092] As used herein and throughout the description, the terms "aryl" or "aromatic ring," as used herein, refer to an aromatic monocyclic or polycyclic system having 3 to 20 ring atoms, wherein all ring atoms are carbon and may be substituted or unsubstituted. In certain specific embodiments of the invention, "aryl" relates to a monocyclic, bicyclic, or tricyclic C4-C20 aromatic ring system having one, two, or three aromatic rings, including but not limited to phenyl, biphenyl, naphthyl, etc., which may have one or more substituents. Preferably, the aryl group contains 3 to 14 (e.g., 5 to 10, such as 5, 6, or 10) carbon atoms, more preferably 6 to 10 carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropylonyl, cyclopentadienyl, phenyl, indenyl, naphthyl, azulel, fumonyl, anthraceneyl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. In some embodiments, the aryl group used in this invention contains 3 to 20 carbon atoms (C3-20 aryl). In some embodiments, the aryl group used in this invention contains 3 to 18 carbon atoms (C3-18 aryl). In another embodiment, the aryl group used in this invention contains 3 to 16 carbon atoms (C3-16 aryl). In another embodiment, the aryl group used in this invention contains 6 to 16 carbon atoms (C6-16 aryl). In another embodiment, the aryl group used in this invention contains 7 to 16 carbon atoms (C7-16 aryl). In another embodiment, the aryl group used in this invention contains 6 to 14 carbon atoms (C6-14 aryl). In yet another embodiment, the aryl group used in this invention contains 7 to 14 carbon atoms (C7-14 aryl). In another specific embodiment, the aryl group used in this invention contains 6 to 12 carbon atoms (C6-12 aryl). In another specific embodiment, the aryl group used in this invention contains 7 to 12 carbon atoms (C7-12 aryl). In another specific embodiment, the aryl group used in this invention contains 6 to 11 carbon atoms (C6-11 aryl). In another specific embodiment, the aryl group used in this invention contains 7 to 11 carbon atoms (C7-11 aryl). In another specific embodiment, the aryl group used in this invention contains 6 to 10 carbon atoms (C6-10 aryl). In another specific embodiment, the aryl group used in this invention contains 7 to 10 carbon atoms (C7-10 aryl). In another specific embodiment, the aryl group used in this invention contains 6 to 8 carbon atoms (C6-8 aryl). In another specific embodiment, the aryl group used in this invention contains 6 carbon atoms (C6 aryl). In another specific embodiment, the aryl group used in this invention contains 10 carbon atoms (C10 aryl). In some specific embodiments, Z is neither a substituted monocyclic six-membered aryl group nor an unsubstituted monocyclic six-membered aryl group.In some specific embodiments, Z is not a substituted phenyl or an unsubstituted phenyl. In some specific embodiments, Z is not a phenyl substituted with 1, 2, 3, 4, or 5 substituents selected from –NO2, -N3, halogen, -NH2, hydroxyl, -OR11, and –C(=O)R11, wherein R11 is hydrogen, a substituted alkyl, or a substituted alkynyl. Aryl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0093] As used herein and throughout the specification, the term "aryl" as used herein refers to an aryl diradical derived from an aryl group as defined herein by removing two hydrogen atoms. The aryl group may be substituted or unsubstituted. Aryl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety. Additionally, the aryl group may be incorporated as a linking group into arylalkyl, arylenyl, arylynyl, arylhexaneyl, arylenyl, or arylhexaneyl groups as defined herein.

[0094] As used herein and throughout the specification, the terms "heteroaryl" or "heteroary ring" refer to the aryl group as defined above, wherein one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, the heteroaryl group relates to a five- or six-membered aromatic monocyclic ring, wherein one, two, or three carbon atoms are replaced by the same or different O, N, or S heteroatoms. Alternatively, it means an aromatic bicyclic or tricyclic system, wherein one, two, three, four, or five carbon atoms are replaced by the same or different O, N, or S heteroatoms. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is one, the maximum number of S atoms is one, and the maximum total number of O and S atoms is two. In some specific embodiments, the heteroaryl group used in this invention is a five-membered aromatic monocyclic ring, wherein one, two, or three carbon atoms are replaced by the same or different O, N, or S heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a five-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different O heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a five-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different O and N heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a five-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different O and S heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a five-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different N and S heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a six-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different O, S, or N heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is a six-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by N. In some specific embodiments, the heteroaryl group used in this invention is an aromatic bicyclic system, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different O, N, or S heteroatoms. In some specific embodiments, the heteroaryl group used in this invention is an aromatic bicyclic system, wherein one carbon atom is replaced by O. In some specific embodiments, the heteroaryl group used in this invention is an aromatic bicyclic system, wherein one carbon atom is replaced by N. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted indole group. In some specific embodiments, the heteroaryl group used in this invention is an aromatic bicyclic system, wherein two carbon atoms are replaced by N. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted 7-azaindole group. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted 6-azaindole group. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted 5-azaindole group. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted 4-azaindole group. In some specific embodiments, the heteroaryl group is a substituted or unsubstituted imidazolyl group. In some specific embodiments, the heteroaryl group used in this invention is an aromatic bicyclic system in which three carbon atoms are replaced by N, preferably a substituted or unsubstituted diazaindolyl group.Exemplary heteroaryl groups include furanyl, thiophene, acezolyl, isozolyl, acediazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyridine, triazolyl (1... ,2,3-, 1,2,4- and 1,3,5-), benzofuranyl (1- and 2-), indoleyl, azaindoleyl (4-, 5-, 6- and 7-), diazaindoleyl, isoindoleyl, benzothiopheneyl (1- and 2-), 1H-indazoleyl, benzimidazolyl, benzoxazolyl, indoleyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzo[…] Triazolyl, quinolinyl, isoquinolinyl, benzodiazolyl, quinoxalinyl, quinazolinyl, benzotriazolyl (1,2,3- and 1,2,4-benzotriazolyl), pyridyl, phenazolyl, thiazopyridyl, pyrrolothiazolyl, phenthiazolyl, isobenzofuranyl, chromenyl, syl, phenazolyl, pyrrololyl, indyl, indazolyl, purinyl, quinolinyl, tanyl, tanyl, tanyl Phenylacetic substituents (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), phenolinyl, pteridinyl, carbazolyl, phenidinyl, acridineyl, teridineyl, phenolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), phenoyl, terazolopyridyl, isoterazolopyridyl, pyrroloterazolyl, pyrrolopyrroleyl, etc., having one or more substituents. Heteroaryl substituents include, but are not limited to, any substituents described herein, which result in the formation of a stable moiety. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophene, acezolyl, isoacezolyl, acediazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyracloyl, triracloyl (1,2,3-, 1,2,4- and 1,3,5-), and pyridyl. Exemplary bicyclic heteroaryl groups are 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl, diazaindolyl, and indolyl.

[0095] As used herein and throughout the specification, the terms "diazaindolyl" or "diazaindole" refer to compounds having an indole core structure, wherein two carbon atoms of the cyclic benzene ring are replaced by N. Preferably, 4, 5, 6, and / or 7 carbon atoms of the indole core are replaced by N. Preferably, 4 and 5 carbon atoms of the indole core are replaced by N. Preferably, 4 and 6 carbon atoms of the indole core are replaced by N. Preferably, 4 and 7 carbon atoms of the indole core are replaced by N. Preferably, 5 and 6 carbon atoms of the indole core are replaced by N. Preferably, 6 and 7 carbon atoms of the indole core are replaced by N. Preferably, 5 and 7 carbon atoms of the indole core are replaced by N. In some specific embodiments, the diazaindolyl group is substituted. In some specific embodiments, the diazaindolyl group is unsubstituted.

[0096] As used herein and throughout the specification, the term "heteroaryl" as used herein refers to a diradical derived from a heteroaryl group as defined herein by removing two hydrogen atoms. Heteroaryl groups may be substituted or unsubstituted. Additionally, heteroaryl groups may be incorporated as linking groups into heteroalkyl, heterenoyl, heteroynyl, heteroalkyl, heterenoyl, or heteroynyl groups as defined herein. Heteroaryl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0097] As used herein and throughout the specification, the terms "arylene" and "heteroarylene" include groups in which aryl and heteroaryl groups are respectively linked to alkyl groups (e.g., benzyl, phenethyl, pyridinemethyl, etc.), such alkyl groups including alkyl groups in which a carbon atom (e.g., methylene) is replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridoxymethyl, 3-(1-naphthoxy)propyl, etc.). Preferably, the arylene is a substituted or unsubstituted (C6-C14)aryl (C1-C6) alkyl. Preferably, the arylene is a substituted or unsubstituted (C6-C10)aryl (C1-C6) alkyl. Preferably, the heteroarylene is a substituted or unsubstituted (C3-C14)heteroaryl (C1-C6) alkyl. Preferably, the heteroarylene is a substituted or unsubstituted (C3-C10)heteroaryl (C1-C6) alkyl. In some specific embodiments, the alkyl chain is straight-chain. In some embodiments, the alkyl chain is branched. In some embodiments, the alkyl chain is substituted. In some embodiments, the alkyl chain is unsubstituted. In some embodiments, the alkyl chain is straight-chain and is either substituted or unsubstituted. In some embodiments, the alkyl chain is branched and is either substituted or unsubstituted.

[0098] As used herein and throughout the specification, the terms "heterocyclic group," "heterocyclic," or "heterocyclic" refer to cyclic heteroaliphatic groups. Heterocyclic groups refer to non-aromatic, partially unsaturated, or fully saturated 3- to 10-membered ring systems, including monocyclic systems of 3 to 8 atoms, and bicyclic and tricyclic systems, which may include aromatic five- or six-membered aryl or heteroaryl groups fused with a non-aromatic ring. Heterocyclic groups may be substituted or unsubstituted. These heterocycles include those having one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In some specific embodiments, the term "heterocyclic" refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group, wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may optionally be oxidized), and the remaining ring atoms are carbon, the group being connected to the rest of the molecule via any ring atom. Heterocyclic groups include, but are not limited to, bicyclic or tricyclic groups comprising fused 5-, 6-, or 7-membered rings having one to three independently selected heteroatoms from oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatoms may optionally be quaternized, and (iv) any of the above heterocycles may be fused with an aryl or heteroaryl ring. Preferably, in each ring of the heterocyclic group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclic group" also covers the partially or fully hydrogenated forms of the aforementioned heteroaryl groups (such as dihydrogen, tetrahydrogen, or all-hydrogen forms).Exemplary heterocyclic groups include pyrrolidyl, isopyrrolidyl, pyrrolidyl, pyrazoleidyl, pyrazoleidyl, piperidinyl, piperidine, indololinyl, isoindololinyl, di- and tetrahydrofuranyl, di- and tetrahydrothiophenyl, di- and tetrahydroisoazolyl, di- and tetrahydroisoazolyl, di- and tetrahydroisodiazolyl (1,2,5- and 1,2,3-), and dihydropyrrolidyl Pyroyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydrothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyridine, di- and tetrahydrotriazolyl (1,2,3-) -, 1,2,4- and 1,3,5-), di-tetrahydrobenzofuranyl (1- and 2-), di-tetrahydroindolyl, di-tetrahydroisoindolyl, di-tetrahydrobenzothiophenyl (1- and 2-), di-tetrahydro-1H-indazolyl, di-tetrahydrobenzimidazolyl, di-tetrahydrobenzoxazolyl, di-tetrahydroindolyl, di-tetrahydrobenzisisoxazolyl, di-tetrahydrobenzisisothiazolyl, di-tetrahydrobenzisisothiazolyl, di-tetrahydrobenzistriazolyl, di-tetrahydroquinolinyl, di-tetrahydroisoquinolinyl, di-tetrahydrobenzodiazolyl, di-tetrahydroquinoxolinyl, di-tetrahydroquinoxolinyl, di-tetrahydrobenzotriazolyl (1,2,3- and 1,2,4-), di-tetrahydropyridyl, Di- and tetrahydrophenanthrylyl, di- and tetrahydrothiazopyridyl (such as 4,5,6-7-tetrahydro[1,3]thiazo[5,4-c]pyridyl or 4,5,6-7-tetrahydro[1,3]thiazo[4,5-c]pyridyl, for example 4,5,6-7-tetrahydro[1,3]thiazo[5,4-c]pyridin-2-yl or 4,5,6-7-tetrahydro[1,3]thiazo[4,5-c]pyridin-2-yl), di- and tetrahydropyrrolothiazolyl (such as 5,6-dihydro-4H-pyrrolo[3,4-d][1,3]thiazolyl), di- and tetrahydrophenanthrylyl, di- and tetrahydroisobenzofuranyl, di- and tetrahydrophenenylyl, di- and tetrahydrophenylylylyl, di- and tetrahydropyrrolyl ... Hydrophenanthrethioyl, di- and tetrahydropyrrolidinyl, di- and tetrahydroindoleyl, di- and tetrahydroindazoleyl, di- and tetrahydropurinyl, di- and tetrahydroquinoyl, di- and tetrahydrocarbazoleyl, di- and tetrahydropyridyl (1,5-, 1,6-, 1,7, 1,8- and 2,6-), di- and tetrahydropyridineyl, di- and tetrahydrocarbazoleyl, Di-tetrahydrophenidyl, di-tetrahydroacridyl, di-tetrahydropyridyl, di-tetrahydrophenolinyl (1,7-, 1,8-, 1,10-, 3,8- and 4,7-), di-tetrahydrophenidyl, di-tetrahydropyrazolyl, di-tetrahydroisopyrazolyl, di-tetrahydropyrrolopyrrolyl, and di-tetrahydropyrrolopyrroleyl.Exemplary 5- or 6-membered heterocyclic groups include pyrrolidyl, pyrrolidyl, imidazolidyl, pyrazolidyl, piperidinyl, piperidine, di- and tetrahydrofuranyl, di- and tetrahydrothiophenyl, di- and tetrahydroisoazolyl, di- and tetrahydrodiazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,5- and 1,2,3-), and 2- and 3-triazolyl (1,2,5- and 1,2,3-). 3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydroisothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyridine, di- and tetrahydrotriazolyl (1,2,3-, 1,2,4- and 1,3,5-), di- and tetrahydropyridyl, etc., which may have one or more substituents. Preferred heterocyclic groups are 2H-1-benzopyranyl (2H-alkyl), benzodihydropyranyl (alkyl), 4H-1-benzopyranyl (4H-alkyl), 1H-2-benzopyranyl (1H-isoalkyl), isoalkyl, 3H-2-benzopyranyl (3H-isoalkyl), 1-benzopyran-4-one-yl (chromone-yl), 4-alkyl, 1-benzopyran-2-one-yl (coumarinyl), dihydrocoumarinyl, 3-isoalkylone-yl, and 2-coumaranonyl. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 2H-1-benzopyranyl (2H-alkyl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted benzodihydropyranyl (α-yl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 4H-1-benzopyranyl (4H-α-enyl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 1H-2-benzopyranyl (1H-iso-α-enyl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted iso-α-yl. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 3H-2-benzopyranyl (3H-iso-α-enyl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 1-benzopyran-4-one-yl (chromone-yl). In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 4-α-yl. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl). In some embodiments, the heterocyclic group is a substituted or unsubstituted dihydrocoumarinyl group. In some embodiments, the heterocyclic group is a substituted or unsubstituted 3-isocarboxyl group. In some embodiments, the heterocyclic group is a substituted or unsubstituted 2-coumarinyl group. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C3-C14) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different O, N, or S heteroatoms. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C3-C14) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by O.In some specific embodiments, the heterocyclic group is a substituted or unsubstituted (C3-C14) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by N. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted (C3-C14) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by S. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted (C9-C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different O, N, or S heteroatoms. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted (C9-C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by O. In some specific embodiments, the heterocyclic group is a substituted or unsubstituted (C9-C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by N. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C9-C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by S. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different O, N, or S heteroatoms. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by O. In some embodiments, the heterocyclic group is a substituted or unsubstituted (C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by N. In some embodiments, the heterocyclic group is a substituent-unsubstituted or unsubstituted (C10) heterocyclic group, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by S.

[0099] As used herein and throughout the specification, the term "heteroalkyl" as used herein refers to an alkyl portion as defined herein, containing one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between carbon atoms. Heteroalkyl groups may be substituted or unsubstituted. In some embodiments, a heteroalkyl group contains 1 to 20 carbon atoms and 1 to 6 heteroatoms (C1-20 heteroalkyl). In some embodiments, a heteroalkyl group contains 1 to 10 carbon atoms and 1 to 4 heteroatoms (C1-10 heteroalkyl). In some embodiments, a heteroalkyl group contains 1 to 6 carbon atoms and 1 to 3 heteroatoms (C1-6 heteroalkyl). In some embodiments, a heteroalkyl group contains 1 to 5 carbon atoms and 1 to 3 heteroatoms (C1-5 heteroalkyl). In some embodiments, a heteroalkyl group contains 1 to 4 carbon atoms and 1 to 2 heteroatoms (C1-4 heteroalkyl). In some embodiments, a heteroalkyl group contains 1 to 3 carbon atoms and 1 heteroatomum (C1-3 heteroalkyl). In some specific embodiments, the heteroalkyl group contains 1 to 2 carbon atoms and 1 heteroatom (C1-2 heteroalkyl). As used herein, the term "heteroalkyl" refers to a diradical derived from a heteroalkyl group as defined herein by removing two hydrogen atoms. Heteroalkyl groups can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In some specific embodiments, the heteroalkyl group is a substituted heteroalkyl group (C1-6 heteroalkyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms. In some specific embodiments, the heteroalkyl group is an unsubstituted heteroalkyl group (C1-6 heteroalkyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms. In some specific embodiments, the heteroalkyl group is an alkyl moiety in which one methylene group is replaced by S. In some specific embodiments, the heteroalkyl group is an alkyl moiety in which one methylene group is replaced by O. In some specific embodiments, the heteroalkyl group is an alkyl moiety in which one methylene group is replaced by NR1, wherein the moiety is selected from the group consisting of: hydrogen, substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)ynyl, substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted (C6-C14)aryl, and substituted or unsubstituted (C3-C14) heteroaryl. In some specific embodiments, the heteroalkyl group is –CH2SCH3. In some specific embodiments, the heteroalkyl group is –CH2OCH3.

[0100] As used herein and throughout the description, the alkyl substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0101] As used herein and throughout the specification, the term "heteroalkenyl" as used herein refers to an alkenyl portion as defined herein, which further contains one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between carbon atoms. In some embodiments, the heteroalkenyl contains 2 to 20 carbon atoms and 1 to 6 heteroatoms (C2-20 heteroalkenyl). In some embodiments, the heteroalkenyl contains 2 to 10 carbon atoms and 1 to 4 heteroatoms (C2-10 heteroalkenyl). In some embodiments, the heteroalkenyl contains 2 to 6 carbon atoms and 1 to 3 heteroatoms (C2-6 heteroalkenyl). In some embodiments, the heteroalkenyl contains 2 to 5 carbon atoms and 1 to 3 heteroatoms (C2-5 heteroalkenyl). In some embodiments, the heteroalkenyl contains 2 to 4 carbon atoms and 1 to 2 heteroatoms (C2-4 heteroalkenyl). In some specific embodiments, the heteroalkenyl group contains 2 to 3 carbon atoms and 1 heteroatom (C2-3 heteroalkenyl). As used herein, the term "hexalkenyl" refers to a diradical derived from a heteroalkenyl group as defined herein by removing two hydrogen atoms. The hexalkenyl group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In some specific embodiments, the hexalkenyl group is a substituted hexalkenyl group (C1-6 hexalkenyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms. In some specific embodiments, the hexalkenyl group is an unsubstituted hexalkenyl group (C1-6 hexalkenyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms.

[0102] As used herein and throughout the specification, the term "pyrynyl" as used herein refers to the yynyl group as defined herein, which further contains one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between carbon atoms. In some embodiments, the pyrynyl group contains 2 to 20 carbon atoms and 1 to 6 heteroatoms (C2-20 pyrynyl). In some embodiments, the pyrynyl group contains 2 to 10 carbon atoms and 1 to 4 heteroatoms (C2-10 pyrynyl). In some embodiments, the pyrynyl group contains 2 to 6 carbon atoms and 1 to 3 heteroatoms (C2-6 pyrynyl). In some embodiments, the pyrynyl group contains 2 to 5 carbon atoms and 1 to 3 heteroatoms (C2-5 pyrynyl). In some embodiments, the pyrynyl group contains 2 to 4 carbon atoms and 1 to 2 heteroatoms (C2-4 pyrynyl). In some specific embodiments, the cycloynyl group contains 2 to 3 carbon atoms and 1 heteroatom (C2-3 cycloynyl). As used herein, the term "cycloynyl" refers to a diradical derived from a cycloynyl group as defined herein by removing two hydrogen atoms. The cycloynyl group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In some specific embodiments, the cycloynyl group is a substituted cycloynyl group (C1-6 cycloynyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms. In some specific embodiments, the cycloynyl group is an unsubstituted cycloynyl group (C1-6 cycloynyl) containing 1 to 6 carbon atoms and 1 to 3 heteroatoms.

[0103] The non-natural amino acids described herein can also be described by having Formula I: (I)

[0104] In some specific embodiments, the compounds according to Formula I are characterized in that X is O, NR1, or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; and Z is selected from the group consisting of: substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C2-C6) alkenyl, substituted or unsubstituted (C2-C6) alkynyl, substituted or unsubstituted (C3-C8) cycloalkyl, substituted or unsubstituted (C6-C14) aryl, substituted or unsubstituted (C6-C14) alkyl, etc. 4) aryl (C1-C6) alkyl, substituted or unsubstituted (C3-C14) heteroaryl, substituted or unsubstituted (C3-C14) heteroaryl (C1-C6) alkyl, substituted or unsubstituted (C3-C14) heterocyclic, substituted or unsubstituted (C1-C6) heteroalkyl, substituted or unsubstituted (C2-C6) heteroalkenyl, and substituted or unsubstituted (C2-C6) heteroynyl; R1 is hydrogen or substituted or unsubstituted (C1-C6) alkyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0105] In some specific embodiments, the compound according to Formula I is characterized in that X is O, NR1 or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl; Z is selected from the group consisting of: substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C6-C14) aryl, substituted or unsubstituted (C3-C14) heteroaryl, substituted or unsubstituted (C3-C14) heterocyclic, and substituted or unsubstituted (C1-C6) heteroalkyl; R1 is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0106] In some specific embodiments, the compound according to Formula I is characterized in that X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl; Z is selected from the group consisting of: substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C6-C14) aryl, substituted or unsubstituted (C3-C14) heteroaryl, substituted or unsubstituted (C3-C14) heterocyclic, and substituted or unsubstituted (C1-C6) heteroalkyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0107] In some specific embodiments, the compound according to formula I is characterized in that X is O, NR1 or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; Z is selected from the group consisting of substituted or unsubstituted (C6-C14) aryl, substituted or unsubstituted (C3-C14) heteroaryl, and substituted or unsubstituted (C3-C14) heterocyclic group; R1 is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0108] In some specific embodiments, the compound according to formula I is characterized in that X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; Z is selected from the group consisting of substituted or unsubstituted (C6-C14) aryl, substituted or unsubstituted (C3-C14) heteroaryl, and substituted or unsubstituted (C3-C14) heterocyclic group, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0109] In some specific embodiments, the compound according to Formula I is characterized in that X is O, NR1, or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; Z is selected from the group consisting of: substituted or unsubstituted 2H-1-benzopyranyl (2H-benzoenyl), substituted or unsubstituted benzodihydropyranyl (benzoyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-benzoenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isobenzoenyl), substituted or unsubstituted isobenzoyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isobenzoenyl), substituted or unsubstituted 1-benzopyran-4-one-yl (chromone-yl), etc. The substituted or unsubstituted 4-carboxyl, substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted 3-isocarboxyl, substituted or unsubstituted 2-coumarinyl, substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, substituted or unsubstituted diazaindolyl, and substituted or unsubstituted indolyl; R1 is hydrogen or substituted or unsubstituted (C1-C6)alkyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0110] In some specific embodiments, the compound according to Formula I is characterized in that X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; and Z is selected from the group consisting of: substituted or unsubstituted 2H-1-benzopyranyl (2H-alkyl), substituted or unsubstituted benzodihydropyranyl (alkyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-alkyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isoalkyl), substituted or unsubstituted isoalkyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isoalkyl), substituted or unsubstituted 1-benzopyran-4-one- The compound comprises: 4-benzopyran-2-one-coumarin, substituted or unsubstituted 4-benzopyran-2-one-coumarin, substituted or unsubstituted dihydrocoumarin, substituted or unsubstituted 3-isobenzopyran, substituted or unsubstituted 2-coumarinone, substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, substituted or unsubstituted diazaindolyl, and substituted or unsubstituted indolyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0111] In some specific embodiments, the compound according to Formula I is characterized in that X is O, NR1, or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; Z is selected from the group consisting of: substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, and 4-azaindolyl, and substituted or unsubstituted indolyl; R1 is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0112] In some specific embodiments, the compound according to Formula I is characterized in that X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group; Z is selected from the group consisting of: substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, and substituted or unsubstituted indolyl, wherein A is methylene and n is 0 or 1, preferably n is 1.

[0113] In some specific embodiments of compounds having the chemical formula according to Formula I, X is O. In some specific embodiments of compounds having the chemical formula according to Formula I, X is S. In some specific embodiments of compounds having the chemical formula according to Formula I, X is NR1, wherein R1 is selected from the group consisting of: hydrogen, substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)ynyl, substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted (C6-C14)aryl, and substituted or unsubstituted (C3-C14)heteroaryl, preferably hydrogen or substituted or unsubstituted (C1-C6)alkyl, more preferably hydrogen, more preferably substituted or unsubstituted (C1-C6)alkyl, and even more preferably unsubstituted (C1-C6)alkyl.

[0114] In some specific embodiments of compounds having the chemical formula according to Formula I, Y is selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, and substituted or unsubstituted heteroalkyl. In some specific embodiments of compounds having a chemical formula according to Formula I, Y is selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)ynyl, substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted (C6-C14)aryl, substituted or unsubstituted (C6-C14)aryl(C1-C6)alkyl, substituted or unsubstituted (C3-C14)heteroaryl, substituted or unsubstituted (C3-C14)heteroaryl(C1-C6)alkyl, substituted or unsubstituted (C3-C14)heterocyclic, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted (C2-C6)heterenyl, and substituted or unsubstituted (C2-C6)heterynyl. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is hydrogen. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted alkyl group, preferably a substituted or unsubstituted (C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted alkyne group, preferably a substituted or unsubstituted (C2-C6) alkyne group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted cycloalkyl group, preferably a substituted or unsubstituted (C3-C8) cycloalkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted aryl group, preferably a substituted or unsubstituted (C6-C14) aryl group.In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted aralkyl group, preferably a substituted or unsubstituted (C6-C14)aryl(C1-C6)alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroaryl group, preferably a substituted or unsubstituted (C3-C14)heteroaryl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroalkyl group, preferably a substituted or unsubstituted (C6-C14)heteroaryl(C1-C6)alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroalkyl group, preferably a substituted or unsubstituted (C3-C14)heteroalkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroalkyl group, preferably a substituted or unsubstituted (C1-C6)heteroalkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroenyl group, preferably a substituted or unsubstituted (C2-C6) heteroenyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Y is a substituted or unsubstituted heteroynyl group, preferably a substituted or unsubstituted (C2-C6) heteroynyl group.

[0115] In some specific embodiments of compounds having the chemical formula according to Formula I, Z is selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; provided that Z is not a substituted or unsubstituted monocyclic six-membered aryl. In some specific embodiments of compounds having a chemical formula according to Formula I, Z is selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloyl, and substituted or unsubstituted heteroalkyl; provided that Z is not a substituted or unsubstituted monocyclic six-membered aryl. In some specific embodiments of compounds having a chemical formula according to Formula I, Z is selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)ynyl, substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted (C6-C14)aryl, substituted or unsubstituted (C6-C14)aryl(C1-C6)alkyl, substituted or unsubstituted (C3-C14)heteroaryl, substituted or unsubstituted (C3-C14)heteroaryl(C1-C6)alkyl, substituted or unsubstituted (C3-C14)heterocyclic, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted (C2-C6)heterenyl, and substituted or unsubstituted (C2-C6)heterynyl; provided that Z is not a substituted or unsubstituted monocyclic six-membered aryl. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is hydrogen or a substituted or unsubstituted (C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is hydrogen. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted alkyl group, preferably a substituted or unsubstituted (C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted alkyne group, preferably a substituted or unsubstituted (C2-C6) alkyne group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted cycloalkyl group, preferably a substituted or unsubstituted (C3-C8) cycloalkyl group.In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted aryl group, preferably a substituted or unsubstituted (C6-C14) aryl group, and more preferably Z is a non-substituted or unsubstituted monocyclic six-membered aryl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted aralkyl group, preferably a substituted or unsubstituted (C6-C14) aryl(C1-C6) alkyl group, and more preferably Z is a non-substituted or unsubstituted monocyclic six-membered aryl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heteroaryl group, preferably a substituted or unsubstituted (C3-C14) heteroaryl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heteroarylalkyl group, preferably a substituted or unsubstituted (C3-C14) heteroaryl(C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heterocyclic group, preferably a substituted or unsubstituted (C3-C14) heterocyclic group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heteroalkyl group, preferably a substituted or unsubstituted (C1-C6) heteroalkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heteroalkenyl group, preferably a substituted or unsubstituted (C2-C6) heteroalkenyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted heteroynyl group, preferably a substituted or unsubstituted (C2-C6) heteroynyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is selected from the group consisting of: substituted or unsubstituted 2H-1-benzopyranyl (2H-alkylene), substituted or unsubstituted benzodihydropyranyl (alkyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-alkylene), substituted or unsubstituted 1H-2-benzopyranyl (1H-isoalkylene), substituted or unsubstituted isoalkyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isoalkylene), substituted or unsubstituted 1-benzopyran-4-one- The compounds may contain substituted or unsubstituted 4-benzopyranyl, substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted 3-isobenzoyl, substituted or unsubstituted 2-coumarinyl, substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted imidazoyl, substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, and substituted or unsubstituted indolyl. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is substituted or unsubstituted 2H-1-benzopyranyl (2H-benzoenyl).In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted benzodihydropyranyl (α-yl). In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 4H-1-benzopyranyl (4H-α-enyl). In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 1H-2-benzopyranyl (1H-iso-α-enyl). In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted iso-α-yl. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 3H-2-benzopyranyl (3H-iso-α-enyl). In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 1-benzopyran-4-one-yl (chromone-yl). In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 4-carboxylic acid group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl) group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted dihydrocoumarinyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 3-isocarboxylic acid group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted 2-coumarinyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted (C1-C6) alkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted (C1-C6) heteroalkyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted imidazolylic acid group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted azaindolyl group, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, and 4-azaindolyl. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is a substituted or unsubstituted indolyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is not a substituted monocyclic six-membered aryl group or an unsubstituted monocyclic six-membered aryl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is not a substituted phenyl group or an unsubstituted phenyl group. In some specific embodiments of compounds having the chemical formula according to Formula I, Z is not a phenyl group substituted with 1, 2, 3, 4, or 5 substituents selected from –NO2, -N3, halogen, -NH2, hydroxyl, -OR11, and –C(=O)R11, wherein R11 is hydrogen, a substituted alkyl group, or a substituted alkynyl group.

[0116] In some specific embodiments of compounds having the chemical formula according to Formula I, A is methylene and n is 1. In some specific embodiments of compounds having the chemical formula according to Formula I, A is methylene and n is 2.

[0117] As used herein and throughout the specification, the term "aliphatic" includes saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, optionally substituted with one or more functional groups. As will be understood by those skilled in the art, "aliphatic" herein is intended to include, but is not limited to, alkyl, alkenyl, ynyl, cycloalkyl, cycloalkenyl, and cycloynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched, and cyclic alkyl groups. Similar convention applies to other general terms such as "alkenyl," "ynyl," etc. Additionally, as used herein, the terms "alkyl," "alkenyl," "ynyl," etc., encompass substituted and unsubstituted groups. In some specific embodiments, as used herein, "aliphatic" is used to denote those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 20 carbon atoms (C1-20 aliphatic). In some embodiments, the aliphatic group has 1 to 10 carbon atoms (C1-10 aliphatic). In some embodiments, the aliphatic group has 1 to 6 carbon atoms (C1-6 aliphatic). In some embodiments, the aliphatic group has 1 to 5 carbon atoms (C1-5 aliphatic). In some embodiments, the aliphatic group has 1 to 4 carbon atoms (C1-4 aliphatic). In some embodiments, the aliphatic group has 1 to 3 carbon atoms (C1-3 aliphatic). In some embodiments, the aliphatic group has 1 to 2 carbon atoms (C1-2 aliphatic). Substituents in the aliphatic group include, but are not limited to, any substituents described herein, which result in the formation of a stable moiety. In some embodiments, the aliphatic group is a saturated or unsaturated, unbranched or branched alkyl group, preferably (C1-C20) alkyl, more preferably (C1-C10) alkyl, and even more preferably (C1-C6) alkyl.

[0118] As used herein and throughout the specification, the term "heteroaliphatic" refers to an aliphatic portion as defined herein, including saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, optionally substituted with one or more functional groups, and further containing one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between carbon atoms. In some specific embodiments, the heteroaliphatic portion is substituted by replacing one or more hydrogen atoms thereon with one or more substituents independently. As will be understood by those skilled in the art, "heteroaliphatic" herein is intended to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloynyl portions. Therefore, the term "heteroaliphatic" includes the terms "heteroalkyl," "heteroalkenyl," "heterynyl," etc. Additionally, as used herein, the terms "heteroalkyl," "heteroalkenyl," "heterynyl," etc., encompass substituted and unsubstituted groups. In some specific embodiments, as used herein, "heteroaliphatic" is used to refer to those heteroaliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched (C1-20 heteroaliphatic)) having 1 to 20 carbon atoms and 1 to 6 heteroatoms. In some specific embodiments, the heteroaliphatic group contains 1 to 10 carbon atoms and 1 to 4 heteroatoms (C1-10 heteroaliphatic). In some specific embodiments, the heteroaliphatic group contains 1 to 6 carbon atoms and 1 to 3 heteroatoms (C1-6 heteroaliphatic). In some specific embodiments, the heteroaliphatic group contains 1 to 5 carbon atoms and 1 to 3 heteroatoms (C1-5 heteroaliphatic). In some specific embodiments, the heteroaliphatic group contains... The aliphatic group contains 1 to 4 carbon atoms and 1 to 2 heteroatoms (C1-4 heteroaliphatic). In some specific embodiments, the heteroaliphatic group contains 1 to 3 carbon atoms and 1 heteroatomum (C1-3 heteroaliphatic). In some specific embodiments, the heteroaliphatic group contains 1 to 2 carbon atoms and 1 heteroatomum (C1-2 heteroaliphatic). Substituents in the aliphatic group include, but are not limited to, any substituents described herein, which result in the formation of a stable moiety. In some specific embodiments, the aliphatic group is a saturated or unsaturated, unbranched or branched alkyl group, preferably (C1-C20) alkyl, more preferably (C1-C10) alkyl, and even more preferably (C1-C6) alkyl. "L": Linker

[0119] This document discloses an antibody-drug conjugate in which belenutoxine modified as described herein is linked to a drug moiety. According to this document, belenutoxine can be linked to a drug moiety via covalent linkage of a linker. As used herein, a "linker" is any chemical moiety capable of linking an antibody (such as belenutoxine), an antibody fragment (e.g., an antigen-binding fragment), or a functional equivalent to another moiety (such as a drug moiety). In this regard, the "ADC chemical formula" described herein is again referred to: Ab-(L-(D)x)y. Thus, the drug moiety D can be linked to belenutoxine via linker L. L is any chemical moiety capable of linking belenutoxine antibody to drug moiety D. Preferably, linker L links belenutoxine antibody to drug moiety D via a covalent bond. The linker reagent is a bifunctional or multifunctional moiety that can be used to link drug moiety D and belenutoxine antibody to form an antibody-drug conjugate. The antibody-drug conjugate can be prepared using a linker having a reactive functional group for binding drug moiety D and antibody Ab. The terms "linker reagent", "crosslinking reagent", "linker derived from crosslinking reagent", and "linker" are used interchangeably in this document.

[0120] The linker may be sensitive to cleavage (cleavable linker), such as enzymatic cleavage, acid-induced cleavage, photo-induced cleavage, and disulfide bond cleavage. Enzymatic cleavage includes, but is not limited to, protease-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphatase-induced cleavage, and sulfatase-induced cleavage, preferably under conditions that preserve the activity of the compound or antibody. Alternatively, the linker may be substantially resistant to cleavage (e.g., a stable linker or a non-cleavable linker). In some aspects, the linker may be a pre-charged linker, a hydrophilic linker, a PEG-based linker, or a dicarboxylic acid-based linker. Thus, in some specific embodiments of any antibody-drug conjugate disclosed herein, the linker (L) is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, PEG-based linkers, pre-charged linkers, and dicarboxylic acid-based linkers. In some specific embodiments, L is a cleavable linker. In some specific embodiments, L is a non-cleavable linker. In some specific embodiments, L is a linker sensitive to enzymatic cleavage. In some specific embodiments, L is an acid-labile linker, a light-labile linker, a peptidase-cleavable linker, a protease-cleavable linker, an esterase-cleavable linker, a glycosidase-cleavable linker, a phosphatase-cleavable linker, a sulfatase-cleavable linker, a disulfide-reducible linker, a hydrophilic linker, a pre-charged linker, a PEG-based linker, or a dicarboxylic acid-based linker. A peptidase-cleavable linker is preferred. Other preferred linkers can be cleaved by proteases.

[0121] A non-cleavable linker is any chemical component capable of stably covalently linking a drug to an antibody, and does not belong to the category of cleavable linkers described above. Therefore, a non-cleavable linker is essentially resistant to acid-induced cleavage, peptidase-induced cleavage, protease-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Furthermore, non-cleavage refers to the ability of the chemical bonds in or adjacent to the linker to resist cleavage induced by acids, light-labile cleaving agents, peptidases, proteases, esterases, or chemical or physiological compounds that cleave disulfide bonds, without the drug or antibody losing its activity.

[0122] Acid-labile linkers are linkers that can be cleaved at acidic pH. For example, some intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4 to 5) and provide conditions suitable for the cleavage of acid-labile linkers.

[0123] Some linkers can be cleaved by peptidases, i.e., peptidases are peptidases that can be cleaved. In this respect, some peptides are readily cleaved intracellularly or extracellularly; see, for example, Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al., 43 Int. J. Cancer, 677-684 (1989). Peptides are composed of α-amino acids and peptide bonds, which are chemically amide bonds between the carboxyl ester group of one amino acid and the amino group of the second amino acid.

[0124] Some linkers can be cleaved by esterases, i.e., esterase-cleavable linkers. In this respect, some esters can be cleaved by esterases present intracellularly or extracellularly. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters made from simple alcohols (such as aliphatic alcohols and small cyclic alcohols and small aromatic alcohols).

[0125] Precharged linkers are derived from charged crosslinking agents that retain their charge after incorporation into an antibody-drug conjugate. Examples of precharged linkers can be found in US 2009 / 0274713.

[0126] The linker may be cleavable, preferably protease-cleavable, and more preferably cathepsin-cleavable (such as cathepsin B). The linker may contain a valine-citrulline moiety, which can be cleaved by cathepsin (such as cathepsin B). The linker may contain a hydroxyl group and the non-natural amino acid contains a formyl group located ortho-to a hydroxyl group in an aromatic ring (such as 3-methoxytyrosine), wherein the hydroxyl group of the conjugated linker forms an oxime with the formyl group of the non-natural amino acid. The linker may contain a cleavage site. A "cleavage site" is an amino acid sequence that is recognized by and hydrolyzed by a protease or peptidase. Preferably, the cleavage site is the valine-citrulline moiety.

[0127] In one specific embodiment, the connector of the ADC of the present invention has the following formula: -Aa-XX-Ww-Yy, where: -A- is a stretcher unit; a is 0 or 1; where -X- is a second spacer unit; x is an integer independently in the range of 0 to 12; each -W- is an amino acid unit independently; w is an integer independently in the range of 0 to 12; -Y- is a first spacer unit; and y is an integer independently in the range of 0 to 12, preferably 0, 1 or 2.

[0128] When an elongation unit (-A-) is present, it enables the ligand unit, i.e., the non-natural amino acid of belentoxicin contained in the ADC of the present invention, to be linked to an amino acid unit (-W-). In this respect, the non-natural amino acid has a functional group that can form a bond with the functional group of the elongation unit. Useful functional groups that may be present on the non-natural amino acid naturally or through chemical manipulation include, but are not limited to, thiol (-SH), amino, azide, alkynyl, hydroxyl, carboxyl, anomeric hydroxyl of carbohydrates, methyl, and carboxyl. In one aspect, the functional groups of the non-natural amino acid are thiol and amino. Preferably, the functional group of the non-natural amino acid is methyl. The thiol group can be generated by reducing the intramolecular disulfide bond of the ligand. Alternatively, a thiol group can be generated by reacting the amino group of the lysine moiety of a non-natural amino acid with 2-iminothiacyclopentane (Traut's Reagent) or another thiol group generating agent.

[0129] The reactive group of the elongator may contain a reaction site that reacts with a modified carbohydrate (-CHO) group that may be present on a non-natural amino acid. For example, the carbohydrate may be mildly oxidized using a reagent such as sodium periodate, or a non-natural methyltyrosine amino acid may be linked to a ligand using TTL, and the resulting (-CHO) unit may be condensed with an elongator containing a functional group such as acehydrazine, oxime, pyrazolone, thiopyrazolone, primary or secondary amine, hydrazine, thiourea, hydrazide, and arylacehydrazine, such as those described by Kaneko, T. (1991) Bioconjugate Chem 2:133-41 et al. Representative elongator units conjugated with the non-natural amino acid of this specific embodiment are described in square brackets of structures VIa, VIb, and VIc, wherein -W-, -Y-, -D, w, and y are defined as above, and L is the non-natural amino acid of belentoxicin contained in the ADC of the present invention. R17 is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaralkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl.

[0130] The extended subunit may also include one or more of the following: wherein G is selected from -Cl, -Br, -I, -O-methanesulfonyl, and -O-toluenesulfonyl; wherein J is selected from -Cl, -Br, -I, -F, -OH, -ON-butadieneimine, -O(4-nitrophenyl), -O-pentafluorophenyl, -O-tetrafluorophenyl, and -OC(O)-OR18; R18 is –C1-C8 alkyl or –aryl, and wherein R17 is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl. The wavy line represents a covalent bond with an amino acid unit W (if present), a spacer unit W (if no amino acid is present but a spacer unit is present), or a drug moiety (if neither an amino acid unit nor a spacer unit is present).

[0131] The extension subunit may contain or be composed of a structure as shown in Structure 1 before being conjugated with a non-natural amino acid: wherein R may represent a covalent bond with the second spacer subunit X, the amino acid unit W, the first spacer subunit W, or the pharmaceutical portion.

[0132] In the linker as described herein, the elongated subunit may comprise or consist of a structure as shown in or composed of structure 6 or 7 before conjugation with a non-natural amino acid: wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; z is independently an integer in the range of 0 to 12; Wherein X is an optional second spacer unit as defined herein, preferably selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or a polyethylene glycol-based linker, such as PEGi, where i is an integer in the range of 2 to 12, preferably selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer in the range of 0 to 12; wherein Y is an optional first spacer unit as defined herein; y is independently an integer in the range of 0 to 12; wherein W is an optional amino acid unit as defined herein; w is independently an integer in the range of 0 to 12; Furthermore, D is the pharmaceutical part, and preferably, the hydroxylamine of structure 6 or 7 is conjugated with the non-natural amino acid of belentoxicin contained in the ADC of the present invention, thereby forming an oxime after conjugation.

[0133] When an amino acid unit (-W-) is present: if a spacer subunit is present, the extension subunit is connected to the spacer subunit; if a spacer subunit is not present, the extension subunit is connected to the drug portion; and if neither the extension subunit nor the spacer subunit is present, the ligand unit is connected to the drug portion.

[0134] Ww- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. Each -W- unit independently has the chemical formula represented in the following square brackets, where w is an integer in the range of 0 to 12: where R19 is hydrogen, methyl, isopropyl, isobutyl, dibutyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3 NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridinemethyl-, 3-pyridinemethyl-, 4-pyridinemethyl-, phenyl, cyclohexyl.

[0135] The amino acid unit may be cleaved by one or more enzymes (including tumor-associated proteases) to release the drug moiety (-D), which, in one specific embodiment, is protonated upon release in vivo to provide the drug (D). The illustrative Ww unit is represented by formulas (VII) to (IX): wherein R20 and R21 are as follows: wherein R20, R21, and R22 are as follows: wherein R20, R21, R22, and R23 are as follows:

[0136] Other suitable linkers are disclosed in Salomon et al., Mol. Pharmaceutics 2019, 16, 12, 4817–4825, which are incorporated herein by reference.

[0137] Exemplary amino acid units include, but are not limited to, units of formula (VII), wherein: R20 is benzyl and R21 is -(CH2)4NH2; R20 is isopropyl and R21 is -(CH2)4NH2; R20 isopropyl and R21 is -(CH2)3NHCONH2. Another exemplary amino acid unit is a unit of formula (VIII), wherein R20 is benzyl, R21 is benzyl, and R22 is -(CH2)4NH2.

[0138] Useful -Ww- units can be designed and their selectivity optimized for enzymatic cleavage by specific enzymes (e.g., tumor-associated proteases). In one specific embodiment, the -Ww- unit is a unit whose cleavage is catalyzed by cathepsins B, C, and D or fibrinolytic enzymes ("tumor-associated proteases").

[0139] In one specific embodiment, -Ww- is a dipeptide, tripeptide, tetrapeptide or pentapeptide.

[0140] When R19, R20, R21, R22 or R23 is not hydrogen, the carbon atom bonded to R19, R20, R21, R22 or R23 is chiral.

[0141] Each carbon atom connected to R19, R20, R21, R22 or R23 is independently of (S) or (R) configuration.

[0142] In one aspect, the amino acid unit is valine-citrulline. In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In another aspect, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinoline carboxylic acid-lysine, cyclohexylalanine-lysine, isoepecotic lysine, β-alanine-lysine, glycine, serine, valine, glutamine, and isoepecotic.

[0143] In some embodiments, the amino acid unit may comprise a natural amino acid. In some embodiments, the amino acid unit may comprise a non-natural amino acid.

[0144] When an amino acid unit is present, the first spacer subunit (-Y-) (when present) allows the amino acid unit to be connected to the drug portion. Alternatively, when the amino acid unit is absent, the first spacer subunit allows the extension subunit to be connected to the drug portion. When neither the amino acid unit nor the extension subunit is present, the first spacer subunit also allows the drug portion to be connected to the ligand unit. When the second spacer subunit (-X-) is present, the extension subunit can be connected to the amino acid unit. Alternatively, in the absence of an amino acid unit, the second spacer subunit allows the extension subunit to be connected to the first spacer subunit. Alternatively, in the absence of both the first spacer subunit and the amino acid unit, the second spacer subunit also allows the extension subunit to be connected to the drug portion. The two spacer subunits may be the same or different. The disclosure of "spacer subunit" generally applies to both the first and second spacer subunits. The first and second spacer subunits may be the same. The first and second spacer subunits may be different, wherein each is independently selected from the following examples of spacer subunits.

[0145] There are two general types of spacer units: self-immolative and non-self-immolative. A non-self-immolative spacer unit is one in which, after cleavage (partially enzymatic cleavage) of the amino acid unit from the drug-linker-ligand conjugate or drug-linker compound, part or all of the spacer unit remains bound to the drug moiety. Examples of non-self-immolative spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units (both shown in Scheme 1) (hereinafter). When an exemplary compound containing a glycine-glycine spacer unit or a glycine spacer unit is enzymatically cleaved by a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease, the glycine-glycine-drug moiety or glycine-drug moiety is cleaved from L-Aa-Ww-. In one specific embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-drug moiety bond and releasing the drug.

[0146] In another specific embodiment, the spacer subunit is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3), the extended phenyl portion of which is replaced by Qm, wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer in the range of 0 to 4.

[0147] In one specific embodiment, the non-self-consuming spacer subunit is -Gly-Gly-. In another specific embodiment, the non-self-consuming spacer subunit is -Gly-.

[0148] In one embodiment, an ADC is provided in which a first spacer subunit is absent (y = 0) or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, an ADC is provided in which a second spacer subunit is absent (x = 0) or a pharmaceutically acceptable salt or solvate thereof.

[0149] Alternatively, an exemplary compound containing a self-consuming spacer subunit may release -D without requiring a separate hydrolysis step. In this specific embodiment, the spacer subunit is a PAB group connected to -Ww- via the amino nitrogen atom of the PAB group and directly connected to -D via a carbonate, carbamate, or ether group. Without being bound by any particular theory or mechanism, Scheme 2 describes a possible mechanism for drug release of a PAB group directly connected to -D via a carbamate or carbonate group, supported by Toki et al. (2002) J Org. Chem. 67:1866-1872. Wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; and p ranges from 1 to about 20.

[0150] Without being bound by any particular theory or mechanism, Scheme 3 describes a possible mechanism for drug release from a PAB group directly linked to -D via an ether or amine bond. Wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; and p ranges from 1 to about 20.

[0151] Other examples of self-consuming spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazolium-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that cyclize upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94, 5815), and 2-aminophenylpropionate amides (Amsberry et al., J. Org. Chem., 1990, 55, 5867). Removing the amine-containing drug that is substituted at the α-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self-consuming spacer that can be used in exemplary compounds.

[0152] In one specific embodiment, the spacer subunit is a branched bis(hydroxymethyl)styrene (BHMS) unit as shown in Scheme 4, which can be used for incorporation and release of multiple drugs. Wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer in the range of 0 to 4; n is 0 or 1; and p ranges from 1 to about 20.

[0153] In one embodiment, the -D portion is the same. In another embodiment, the -D portion is different.

[0154] In one respect, the spacer subunit is represented by formulas (X) to (XII): where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer in the range of 0 to 4; and.

[0155] The first spacer subunit (-X-) may be selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or polyethylene glycol-based linkers such as PEGi, wherein i is an integer in the range of 2 to 12, preferably selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer in the range of 0 to 12.

[0156] The second spacer subunit (-Y-) may be selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaralkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or polyethylene glycol-based linkers such as PEGi, wherein i is an integer in the range of 2 to 12, preferably selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer in the range of 0 to 12.

[0157] The linker may have or include a structure as shown in Structure 1 before being conjugated with a non-natural amino acid: wherein R is one or more pharmaceutical moieties that are optionally conjugated with the hydroxylamine of Structure 1 via one or more cleavage sites, preferably wherein the hydroxylamine of Structure 1 is conjugated with the non-natural amino acid.

[0158] The linker may comprise or consist of a structure as shown in or composed of structure 6 or 7 before conjugation with a non-natural amino acid: wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocycloyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; z is independently an integer in the range of 0 to 12; Wherein X is an optional second spacer unit as defined herein, preferably selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaralkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl and substituted or unsubstituted heteroalkynyl, or a linker based on polyethylene glycol such as PEGi, where i is an integer in the range of 2 to 12, preferably selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer in the range of 0 to 12; wherein Y is an optional (first) spacer unit as defined herein; y is independently an integer in the range of 0 to 12; Wherein W is an optional amino acid unit as defined herein; w is independently an integer in the range of 0 to 12; and D is the pharmaceutical part, preferably wherein the hydroxylamine of structure 6 or 7 is conjugated with the non-natural amino acid of belentoxicin contained in the ADC of the present invention, thereby forming an oxime after conjugation.

[0159] The linker may have or include a structure as shown in structure 2 or 3 before conjugation with the non-natural amino acid: wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; wherein D is one or more pharmaceutical moieties; and wherein Y is a cleavage site, such as a cleavage site of a cathepsin (such as cathepsin B); preferably wherein the hydroxylamine of structure 2 or 3 is conjugated with the non-natural amino acid.

[0160] The linker may have a structure as shown in structure 4 or 5 before being conjugated with a non-natural amino acid, wherein D is the pharmaceutical part as described herein, preferably MMAE:.

[0161] As used herein, "before conjugation with a non-natural amino acid" describes chemical entities, such as linkers before they form a covalent bond with their counterpart (non-natural amino acid). However, the same applies to linkers or pharmaceutical portions herein, which are not described as covalently conjugated with each other. Those skilled in the art will readily recognize the resulting structures formed by the reaction of the two chemical entities. In the case of structures 1 to 5 as defined herein, the counterpart is a non-natural amino acid containing a formyl group. The non-natural amino acid may be 3-methoxytyrosine, and the hydroxylamine group of the linker having any of the structures 1 to 5 forms an oxime / hydroxyimine with the 3-methoxytyrosine group of the non-natural amino acid. The reaction mechanism is as follows: In this document, a monoclonal antibody corresponds to belentoxicin, and the payload involves a pharmaceutical portion as defined herein, which is conjugated with a non-natural amino acid (here, 3-methoxytyrosine) via a linker as defined herein.

[0162] Suitable crosslinking agents for forming a non-cleavable linker between pharmaceutical part D and belentoxicin antibody are well known in the art, and can form non-cleavable linkers containing sulfur atoms (such as SMCC) or without sulfur atoms. The crosslinking agents for forming a non-cleavable linker between pharmaceutical part D and belentoxicin antibody contain a portion based on a maleic anhydride or a haloacetyl group. As disclosed herein, such non-cleavable linkers are said to be derived from portions based on maleic anhydride or a haloacetyl group.

[0163] Crosslinking agents containing a portion based on a cis-butenylimidyl group include, but are not limited to, N-succinimidyl-4-(cis-butenylimidylmethyl)cyclohexane-1-carboxylate (SMCC), sulfonylsuccinimidyl-4-(N-cis-butenylimidylmethyl)cyclohexane-1-carboxylate (sulfon-SMCC), N-succinimidyl-4-(cis-butenylimidylmethyl)cyclohexane-1-carboxy-(6-acylaminohexanoate), which are "long-chain" analogs of SMCC (LC-SMCC), κ-cis-butenylimidyl undecanoate N-succinimidyl ester (KMUA), γ-cis-butenylimidylbutanoate N-succinimidyl ester (GMBS), ε-cis-butenyl N-Butadiene diimide (EMCS), m-cis-butadiene diimide benzoyl-N-hydroxybutadiene diimide (MBS), N-(α-cis-butadiene diimide acetoxy)-butadiene diimide (AMSA), succinimide-6-(β-cis-butadiene diimide propionic acid) hexanoate (SMPH), N-butadiene diimide-4-(p-succinimide phenyl)-butyrate (SMPB), N-(p-succinimide phenyl) isocyanate (PMIP), and crosslinking agents based on the cis-butadiene diimide group containing polyethylene glycol spacers, such as cis-butadiene diimide-PEG-NHS, which is also referred to herein as MAL-PEG-NHS. These crosslinking agents form non-cleavable linkers derived from the maleic diamide group. Representative structures of maleic diamide-based crosslinking agents are shown below.

[0164] In some specific embodiments, the linker L is derived from N-succinimidyl-4-(cis-butenediamide-methyl)cyclohexane carboxylate (SMCC), sulfosuccinimidyl-4-(N-cis-butenediamide-methyl)cyclohexane-1-carboxylate (sulfon-SMCC), or MAL-PEG-NHS.

[0165] Crosslinking agents containing a haloacetyl group-based moiety include N-succiniminoiodoacetate (SIA), N-succinimino(4-iodoacetyl)aminobenzoate (SIAB), N-succiniminobromoacetate (SBA), and N-succinimino3-(bromoacetylamino)propionate (SBAP). These crosslinking agents form non-cleavable linkers derived from the haloacetyl group-based moiety. Representative structures of haloacetyl group-based crosslinking agents are shown below.

[0166] In some specific embodiments, the linker L is derived from N-succiniminoiodoacetate (SIA) or N-succinimino(4-iodoacetyl)aminobenzoate (SIAB).

[0167] Suitable cross-linking agents for forming a cleavable linker between drug moiety D and belentoxicin antibody are well known in the art. Disulfide-containing linkers are linkers that can be cleaved by disulfide exchange, which can occur under physiological conditions. As disclosed herein, such cleavable linkers are said to be derived from disulfide-based moieties. Suitable disulfide cross-linking agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)valerate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-4-(2-pyridyldithio)2-thio-butyrate (sulfo-SPDB), the structures of which are shown below. These disulfide cross-linking agents form cleavable linkers derived from disulfide-based moieties.

[0168] In some specific embodiments, the linker L is derived from N-succinimino-4-(2-pyridyl dithio)butyrate (SPDB).

[0169] A suitable cross-linking agent for forming a charged linker between the drug portion D and the belentoxicin antibody is called a pre-charged cross-linking agent. In one specific embodiment, the linker L is derived from the pre-charged cross-linking agent CX1-1. The structure of CX1-1 is shown below:

[0170] Other linkers applicable to the belentoxidide drug conjugate disclosed herein are cis-butenidimidohexyl (MC), cis-butenidimidohexyl (MC) having a self-cleaving peptide, cis-butenidimidodiaminopropyl (mDPR) having a self-cleaving peptide, and 4-(N-cis-butenidimidomethyl)-cyclohexane-1-carbonyl (MCC).

[0171] In some specific embodiments, the linker is cis-butenediami-imino-hexylyl (MC). Cis-butenediami-imino-hexylyl has the following structure: wherein the wavy line indicates a covalent connection between the carbonyl carbon atom of the linker and another portion of the belentoxidix drug conjugate, particularly a covalent connection with belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably a covalent connection with the drug moiety (D). As a non-limiting example, in a belentoxidix drug conjugate containing the MC linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the cis-butenediami-imino moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0172] The male-butenediamide-hexyl linker can be derived, for example, from a crosslinking agent, which is an NHS ester having the following structure:

[0173] In some specific embodiments, the linker is 4-(N-cis-butenidimidomethyl)-cyclohexane-1-carbonyl (MCC). The MCC has the following structure: , where the wavy line represents the covalently linked carbonyl carbon atom of the linker to another portion of the belentoxigenic drug conjugate, particularly to belentoxigenic or the antigen-binding fragment (Ab) or the drug portion (D), preferably to belentoxigenic or the antigen-binding fragment (Ab). As a non-limiting example, in a belentoxigenic drug conjugate containing an MCC linker, belentoxigenic or its antigen-binding fragment (Ab) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula, and the drug portion (D) may be covalently linked to the cis-butenidimido portion.

[0174] In some specific embodiments, the linker is a maleic-butenediamide-hexylyl (MC) linker having a self-cleaving peptide. Non-limiting examples of maleic-butenediamide-hexylyl linkers having a self-cleaving peptide are described below.

[0175] In some specific embodiments, the linker is a maleic-diamino-diamino-propionic (mDPR) linker having a self-cleaving peptide. Non-limiting examples of maleic-diamino-diamino-propionic (mDPR) linkers having a self-cleaving peptide are described below.

[0176] In some specific embodiments, the linker (L) of any one of the belentoxicin drug conjugates Ab-(L-(D)x)y described herein is derived from a crosslinking agent selected from the group consisting of: N-succinimino-3-(2-pyridyl dithio)propionate (SPDP), N-succinimino-4-(2-pyridyl dithio)valerate (SPP), N-succinimino-4-(2-pyridyl dithio)butyrate (SPDB), N-succinimino-4-(2-pyridyl dithio)2-thio-butyrate (sulfonyl-SPDB), N-succinimino-iodoacetate (SIA), N-succinimino-(4-iodoacetyl)aminobenzoate (SIAB), maleic anhydride-PEG-NHS, Cis-butenylidene-iminohexyl (MC), cis-butenylidene-iminohexyl (MC) with a self-cleaving peptide, cis-butenylidene-diaminopropionic acid (mDPR) with a self-cleaving peptide, 4-(N-cis-butenylidene-iminomethyl)-cyclohexane-1-carbonyl (MCC), N-butadiene-imino-4-(cis-butenylidene-iminomethyl)cyclohexane-1-carboxylic acid ester (S) MCC), N-sulfobutyldiimide 4-(cis-butenidiminomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazahepta-dec-1-ester (CX1-1). In preferred embodiments, the linker is derived from a crosslinking agent selected from the group consisting of: N-succinimino-4-(2-pyridyldithio)butyrate (SPDB), maleic anisodimino-hexyl (MC), maleic anisodimino-hexyl (MC) having a self-cleaving peptide, maleic anisodimino-diaminopropionic acid (mDPR) having a self-cleaving peptide, 4-(N-maleic anisodimino-methyl)-cyclohexane-1-carbonyl (MCC), and N-succinimino-4-(maleic anisodimino-methyl)cyclohexane-1-carboxylic acid ester (SMCC). Therefore, in some embodiments, the linker is derived from N-succinimino-4-(2-pyridyldithio)butyrate (SPDB).

[0177] In some embodiments, the linker is derived from maleic diiminohexyl (MC). In some embodiments, the linker is derived from maleic diiminohexyl (MC) having a self-cleaving peptide. In some embodiments, the linker is derived from maleic diiminodiaminopropyl (mDPR) having a self-cleaving peptide. In some embodiments, the linker is derived from 4-(N-maleic diiminomethyl)-cyclohexane-1-carbonyl (MCC). In some embodiments, the linker is derived from N-butadiene-4-(maleic diiminomethyl)cyclohexane-1-carboxylic acid ester (SMCC).

[0178] As described above, the linker may be a maleic anisodiminohexyl (MC) having a self-cleaving peptide. In some specific embodiments, the linker having the self-cleaving peptide is selected from the group consisting of: maleic anisodiminohexyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleic anisodiminohexyl-valine-alanine-p-aminobenzyloxycarbonyl (MC-VA-PAB), maleic anisodiminohexyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (MC-KF-PAB), and maleic anisodiminohexyl-valine-lysine-p-aminobenzyloxycarbonyl (MC-VK-PAB). Cis-butenediamide hexanoyl (MC) linkers of self-cleaving peptides are disclosed, for example, in U.S. Patent Application Publication US 2006 / 0074008, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003), the contents of which are incorporated herein by reference.

[0179] In some specific embodiments, the linker having the self-cleaving peptide is maleic anhydride-hexyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB). The MC-VC-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the MC-VC-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula. Linkers and the following cis-butenidiminohexyl linkers containing a p-aminobenzyloxycarbonyl group, which are self-cleaving peptides, can, for example, derive self-crosslinking agents, wherein the p-nitrophenoxy group is linked at the position shown by the wavy line (see, for example, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003)). In the case of the MC-VC-PAB linker, the crosslinking agent can therefore have the following structure: When such a crosslinking agent, for example, reacts with a drug moiety, the p-nitrophenoxy group can be partially replaced by the drug moiety.

[0180] Without being bound by a specific theory, in the MC-VC-PAB linker and the cis-butenidiminohexyl linker containing a self-cleaving peptide with a p-aminobenzyloxycarbonyl group described below, protease cleavage can occur between the peptide moiety and the p-aminobenzyloxycarbonyl moiety (see, for example, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003)). The p-aminobenzyloxycarbonyl moiety can then act as a self-consuming group. In this respect, the term "self-consuming group" refers to a bifunctional chemical moiety capable of covalently linking two spaced-apart chemical moieties together to form a normally stable triplet molecule. If its bond with the first moiety is cleaved, it will spontaneously separate from the second chemical moiety.

[0181] In some specific embodiments, the linker having the self-cleaving peptide is maleic anisodimino-hexyl-valine-alanine-p-aminobenzyloxycarbonyl (MC-VA-PAB). The MC-VA-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the MC-VA-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anisodimino moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0182] In some specific embodiments, the linker having the self-cleaving peptide is maleic anhydride-hexyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (MC-KF-PAB). The MC-KF-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the MC-KF-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0183] In some specific embodiments, the linker having the self-cleaving peptide is maleic anhydride-hexyl-valine-lysine-p-aminobenzyloxycarbonyl (MC-VK-PAB). The MC-VK-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the MC-VK-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0184] As described above, the linker may be a maleic diaminodiaminopropyl (mDPR) having a self-cleaving peptide. Such a linker comprises a maleic diaminodiaminopropyl moiety (mDPR) having the following structure:

[0185] In some specific embodiments, the maleic diaminopropyl (mDPR) linker having a self-cleaving peptide is selected from the group consisting of: maleic diaminopropyl-valine-citrulline-p-aminobenzyloxycarbonyl (mDPR-VC-PAB), maleic diaminopropyl-valine-alanine-p-aminobenzyloxycarbonyl (mDPR-VA-PAB), maleic diaminopropyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (mDPR-KF-PAB), and maleic diaminopropyl-valine-lysine-p-aminobenzyloxycarbonyl (mDPR-VK-PAB). Cis-butenediami-imino-diaminopropyl (mDPR) linkers having self-cleaving peptides are disclosed, for example, in U.S. Patent Application Publication US 2013 / 0309256, the entire contents of which are incorporated herein by reference.

[0186] In some specific embodiments, the linker having the self-cleaving peptide is maleic anisodiaminodiaminopropyl-valine-citrulline-p-aminobenzyloxycarbonyl (mDPR-VC-PAB). The mDPR-VC-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the mDPR-VC-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anisodiamino portion, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula. Without being bound by a specific theory, in the mDPR-VC-PAB linker and the cis-butenidiminodiaminopropyl linker containing a p-aminobenzyloxycarbonyl group and having a self-cleaving peptide as described below, protease cleavage can occur between the peptide moiety and the p-aminobenzyloxycarbonyl moiety. The p-aminobenzyloxycarbonyl moiety can then serve as a self-consuming group.

[0187] In some specific embodiments, the linker having the self-cleaving peptide is maleic anisodiaminodiaminopropyl-valine-alanine-p-aminobenzyloxycarbonyl (mDPR-VA-PAB). The mDPR-VA-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the mDPR-VA-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anisodiamino portion, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0188] In some specific embodiments, the linker having the self-cleaving peptide is maleic anhydride-diaminopropyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (mDPR-KF-PAB). The mDPR-KF-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the mDPR-KF-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0189] In some specific embodiments, the linker having the self-cleaving peptide is maleic anisodiaminodiaminopropyl-valine-lysine-p-aminobenzyloxycarbonyl (mDPR-VK-PAB). The mDPR-VK-PAB linker has the following structure: , where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxix drug conjugate containing the mDPR-VK-PAB linker, belentoxix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anisodiamino portion, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0190] In some specific embodiments, the linker of the belentoxicin drug conjugate disclosed herein comprises a glucuronic acid modifier. For linkers having glucuronic acid modifiers, please see, for example, SC Jeffrey et al., Bioconjugate Chem. 2006, 17, 831-840; RP Lyon et al., Nature Biotechnology, vol. 33, 733-736 (2015); U.S. Patent Application Publication US 2013 / 0309256; and International Patent Application Publication WO 2015 / 057699, the entire contents of which are incorporated herein by reference.

[0191] In some specific embodiments, the linker containing the glucuronic acid modifier may be maleic diaminohexyl glucuronide (MC-G) or maleic diaminodiaminopropyl glucuronide (mDPR-G).

[0192] The cis-butenidimidohexyl-glucuronide (MC-G) linker has the following structure: where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxidix drug conjugate, particularly to belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in a belentoxidix drug conjugate containing the MC-G linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the cis-butenidimido moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0193] The maleic anhydride-diaminopropyl glucuronide (mDPR-G) linker has the following structure: wherein the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxidix drug conjugate, particularly to belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxidix drug conjugate containing the mDPR-G linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0194] In some specific embodiments, the linker comprising the glucuronic acid modifier further comprises a polyethylene glycol modifier. For linkers having both glucuronic acid and polyethylene glycol modifiers, please see, for example, RP Lyon et al., Nature Biotechnology, vol. 33, 733-736 (2015) and International Patent Application Publication WO 2015 / 057699, the entire contents of which are incorporated herein by reference.

[0195] In some specific embodiments, the linker comprising a glucuronic acid modifier and further comprising a polyethylene glycol modifier may be maleic diaminohexyl-glucuronide-polyethylene glycol (MC-G-PEG), maleic diaminodiaminopropyl-glucuronide-polyethylene glycol (mDPR-G-PEG), or maleic diaminopropyl-glucuronide-polyethylene glycol (MP-G-PEG).

[0196] The cis-butenyldiimidohexyl-glucuronide-polyethylene glycol (MC-G-PEG) linker has the following structure: where n is an integer from 6 to 72, 8 to 72, 12 to 72, 10 to 72, 12 to 36 or 38, 6 to 24, or 8 to 24; and where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxidix drug conjugate, particularly to belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxidix drug conjugate containing the MC-G-PEG linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the cis-butenyldiimido moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0197] The maleic anhydride-diaminopropyl-glucuronide-polyethylene glycol (mDPR-G-PEG) linker has the following structure: where n is an integer from 6 to 72, 8 to 72, 10 to 72, 12 to 72, 12 to 36 or 38, 6 to 24, or 8 to 24; and where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxidix drug conjugate, particularly to belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxidix drug conjugate containing the mDPR-G-PEG linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0198] The maleic anhydride-propionic acid-glucuronide-polyethylene glycol (MP-G-PEG) linker has the following structure: where n is an integer from 6 to 72, 8 to 72, 10 to 72, 12 to 72, 12 to 36 or 38, 6 to 24, or 8 to 24; and where the wavy line indicates the covalent connection of the carbonyl carbon atom of the linker to another part of the belentoxidix drug conjugate, particularly to belentoxidix or the antigen-binding fragment (Ab) or the drug moiety (D), preferably to the drug moiety (D). As a non-limiting example, in the belentoxidix drug conjugate containing the MP-G-PEG linker, belentoxidix or its antigen-binding fragment (Ab) may be covalently linked to the maleic anhydride moiety, and the drug moiety (D) may be covalently linked to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0199] Without being bound by a specific theory, the release of the drug from the belentoxidide drug conjugate containing the MC-G, mDPR-G, MC-G-PEG, mDPR-G-PEG or MP-G-PEG linker can be initiated by cleaving the glucuronic acid moiety with glucuronidase (see, for example, SC Jeffrey et al., Bioconjugate Chem. 2006, 17, 831-840).

[0200] In some specific embodiments, the linker is a platinum complex linker. For platinum complex linkers, see, for example, NJ Sijbrandi et al., Cancer Res. 2016, 77(2), 257-267, D. Walboer et al., ChemMedChem 2015, 10, 797-803, or U.S. Patent Application US 2014 / 377174, the entire contents of which are incorporated herein by reference.

[0201] In some specific embodiments, the platinum complex linker is an ethylenediamine platinum (II) linker having the following structure: , wherein R independently represents a halogen atom, such as Cl, Br, F or I, preferably Cl. As a non-limiting example, in a belentoxidine drug conjugate containing a platinum complex linker, a belentoxidine or its antigen-binding fragment (Ab) and a drug moiety (D) may be bound to the platinum complex.

[0202] In some specific embodiments, the platinum complex linker, particularly the ethylenediamine platinum (II) linker, and another portion of the belentoxixine drug conjugate, particularly belentoxixine or the antigen-binding fragment (Ab) or the drug portion (D), preferably the drug portion (D), may be separated by a spacer. Suitable spacers may have different characteristics in length, composition, or cleavability. In some specific embodiments, the spacer is 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(piperidin-4-ylmethyl)urea: wherein the NH2 group of the spacer is bound to another portion of the belentoxixine drug conjugate, particularly to belentoxixine or the antigen-binding fragment (Ab) or to the drug portion (D), preferably to the drug portion (D), and the nitrogen atom of the piperidinyl group is bound to the platinum complex linker. The following structure illustrates a preferred embodiment using such a spacer: wherein the NH2 group is bound to another portion of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or to the drug moiety (D), preferably to the drug moiety (D), and R represents the connection to the other portion of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or to the drug moiety (D), preferably to belentoxix or the antigen-binding fragment (Ab). Preferably, the NH2 group is covalently linked to the drug moiety (D), and the platinum complex linker is bound to the belentoxix or the antigen-binding fragment (Ab), as indicated by R. In some specific embodiments, the spacer is a 4-oxo-4-((piperidin-4-ylmethyl)amino)butyryl having the following structure: where the wavy line indicates the covalent connection of the carbonyl carbon atom of the spacer to another part of the belentoxidine drug conjugate, particularly to belentoxidine or the antigen-binding fragment (Ab) or to the drug moiety (D), preferably to the drug moiety (D), and wherein the nitrogen atom of the piperidinyl group is bonded to the platinum complex linker. The following structure illustrates a preferred embodiment using such a spacer: where the wavy line represents the covalent connection of the carbonyl carbon atom to another portion of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or to the drug moiety (D), preferably to the drug moiety (D), and R represents the connection to another portion of the belentoxix drug conjugate, particularly to belentoxix or the antigen-binding fragment (Ab) or to the drug moiety (D), preferably to belentoxix or the antigen-binding fragment (Ab). Preferably, the carbonyl carbon is covalently connected to the drug moiety (D), as shown by the wavy line, and the platinum complex linker is bound to the belentoxix or the antigen-binding fragment (Ab), as shown by R.

[0203] In some specific embodiments, the linker is a disulfide bond instead of a linker. The term "disulfide bond instead of linker," as used throughout this document, refers to a linker capable of replacing the disulfide bond formed by the two cysteines of belentoxicin (Ab). Therefore, when such a disulfide bond instead of linker is combined with the drug moiety (D), the drug moiety (D) is introduced into the space between the two cysteines of belentoxicin (Ab). For disulfide bond instead of linker, see, for example, MEB Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and International Patent Application WO 2013 / 173393, the entire contents of which are incorporated herein by reference.

[0204] In some specific embodiments, the disulfide bond instead of the linker may be a maleic anhydride linker having the following structure: , where X represents a halogen atom, such as Cl, Br, F or I, preferably Cl or Br, more preferably Br, and where the wavy line indicates the covalent connection of the nitrogen atom to the drug moiety (D). For such a linker, see MEB Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965. A preferred specific embodiment is a dibromo-maleic anhydride having the following structure: , where the wavy line indicates the covalent connection of the drug moiety (D) to the maleic anhydride moiety. One or two bromine atoms can react with a thiol group obtained by reducing the disulfide bond of belentoxicin or the antigen-binding fragment (Ab), for example, as shown in the following reaction scheme: where AA1, AA2, AA3, and AA4 comprise a peptide chain of belentoxicin or the antigen-binding fragment (Ab). AA1, AA2, AA3, and AA4 may or may not be part of a single peptide chain. In a preferred embodiment, the pharmaceutical portion is covalently linked to the maleic diimide portion, as shown in R. Prior to the reaction with dibromomaleimide, the disulfide bond of belentoxicin or the antigen-binding fragment (Ab) is reduced using a reducing agent to obtain a free thiol group. Suitable reducing agents include, for example, dithiothreitol (DTT), sodium dithiosulfinate, sodium thiosulfate, sodium sulfite, or triamcinolone sulfite (TCEP), preferably triamcinolone sulfite (TCEP). Following the reduction step, dibromo-cis-butenidium diimide is added to belentoxigenic (Ab), and the dibromo-cis-butenidium diimide reacts with free thiol groups to form a bridging bond between two thiol groups of the cysteine ​​in one or more peptide chains.

[0205] In some embodiments, the disulfide bond instead of the linker comprises a fragment selected from the group consisting of: , , , , , , , and , where Ab represents the linking of a peptide chain to a belentuximab or antigen-binding fragment, and the wavy line represents the linking of the drug moiety (D) to the belentuximab drug conjugate. For such a linker, see WO 2013 / 173393. Therefore, in some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. In some embodiments, the disulfide bond instead of the linker comprises a fragment. Regarding the linking of the drug moiety (D) indicated by the wavy line, in these embodiments, the drug may be linked to the fragment by an optional spacer. Such spacers can be derived, for example, from ethylene glycol oligomers, such as...

[0206] In some specific embodiments, the linker is a glycolinker. The term "glycolinker" as used throughout this document generally refers to a linker containing monosaccharide or oligosaccharide segments. Glycolinkers are described, for example, in F.S. Ekholm et al., ChemMedChem 2016, 11, 2501-2505, U.S. Patent Application US 2016 / 0820797, U.S. Patent Application US 2016 / 0257764, International Patent Application WO 2016 / 053107, U.S. Patent Application US 2016 / 0106860, and International Patent Application WO 2016 / 001485, the entire contents of which are incorporated herein by reference.

[0207] Therefore, the sugar linkers used herein may comprise a segment –G–, wherein G is a monosaccharide, or a straight-chain or branched oligosaccharide comprising 2 to 20, preferably 2 to 12, more preferably 2 to 10, even more preferably 2 to 8, and most preferably 2 to 6 sugar moieties. The sugar moieties that may be present in the segment –G– are known to those skilled in the art and include, for example, glucose (Glc), galactose (Gal), mannose (Man), trehalose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylglucosamine (GalNAc), N-acetylenic acid (NeuNAc), or sialic acid, xylose (Xyl).

[0208] In some embodiments, the glycolinker may comprise a fragment, wherein the wavy line indicates the connection to the belentoxix or antigen-binding fragment (Ab) and the drug portion (D) of the belentoxix drug conjugate. For example, in some embodiments, a glycolinker derived from galactose may be used, which comprises a fragment. In some embodiments, the fragment may be connected to the belentoxix or antigen-binding fragment (Ab) and / or the drug portion (D) by an optional spacer. For example, a glycolinker comprising a monosaccharide fragment and a spacer may have the following structure: , wherein the wavy line indicates the connection to the belentoxix or antigen-binding fragment (Ab) and the drug portion (D) of the belentoxix drug conjugate. For example, the belentoxix or antigen-binding fragment (Ab) may be connected to the glycolinker at a carbonyl carbon atom, and the drug portion (D) may be connected to the monosaccharide fragment, thereby producing the following structure: , wherein Ab is the belentoxix or antigen-binding fragment, and D is the drug portion. In some specific embodiments, when the monosaccharide fragment is galactose, the glycolinker may have the following structure: , where the wavy line indicates the connection to the belentoxicin or antigen-binding fragment (Ab) and the drug portion (D) of the belentoxicin drug conjugate. For example, the belentoxicin or antigen-binding fragment (Ab) may be linked to the glycolinker at the carbonyl carbon atom, and the drug portion (D) may be linked to the monosaccharide fragment, resulting in the following structure: , where Ab is the belentoxicin or antigen-binding fragment (Ab), and D is the drug portion. These specific embodiments of glycolinkers are described, for example, in FS Ekholm et al., ChemMedChem 2016, 11, 2501-2505.

[0209] In some specific embodiments, the sugar linker has a structure in which the wavy line indicates a link to belentoxigenic or antigen-binding fragment (Ab), wherein Su(A)x is a sugar derivative Su containing the x-functional group A, wherein A is independently selected from the group consisting of: thiol group or its precursor, halogen, sulfonyloxy group, halogenated acetaminophen group, thiol acetaminophen group, and sulfonated hydroxyacetaminophen group, wherein x is 1, 2, 3, or 4, and wherein b is 0 or 1. The sugar derivative Su(A)x is derived from a sugar or sugar derivative Su, such as amino sugar or other derived sugar. Examples of sugars and sugar derivatives include galactose (Gal), mannose (Man), glucose (Glc), N-acetylenic acid (NeuNAc) or sialic acid, and trehalose (Fuc). The sugar derivative Su(A)x is preferably derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), trehalose (Fuc), and N-acetylenic acid (sialic acid Sia or NeuNAc), more preferably from the group consisting of GlcNAc, Glc, Gal, and GalNAc. More preferably, Su(A)x is derived from Gal or GalNac, and most preferably, Su(A)x is derived from GalNAc. The drug moiety (D) of the belentoxidix drug conjugate can be linked to the sugar linker by reacting with functional group A. The drug moiety can be linked to the sugar linker via an optional spacer (such as a spacer containing, for example, a maleic anhydride moiety). Sugar linkers of these specific embodiments are described, for example, in U.S. Patent Application US 2016 / 0280797.

[0210] In some specific embodiments, the sugar linker has a structure in which the wavy line indicates a link to belentoxigenic or antigen-binding fragment (Ab), wherein Su(A)x is a sugar derivative Su containing the x-functional group A, wherein A is independently selected from the group consisting of: thiol group or its precursor, halogen, sulfonyloxy group, halogenated acetaminophen group, thiol acetaminophen group, and sulfonated hydroxyacetaminophen group, wherein x is 1, 2, 3, or 4, wherein b is 0 or 1, wherein d is 0 or 1, wherein e is 0 or 1, and wherein G is a monosaccharide or a linear or branched oligosaccharide containing 2 to 20 sugar moieties. The sugar derivative Su(A)x is derived from a sugar or sugar derivative Su, such as an amino sugar or other derived sugar. Examples of sugars and sugar derivatives include galactose (Gal), mannose (Man), glucose (Glc), N-acetylenic acid (NeuNAc) or sialic acid, and trehalose (Fuc). The sugar derivative Su(A)x is preferably derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), trehalose (Fuc), and N-acetylenic acid (sialic acid Sia or NeuNAc), preferably from the group consisting of GlcNAc, Glc, Gal, and GalNAc. More preferably, Su(A)x is derived from Gal or GalNac, and most preferably, Su(A)x is derived from GalNAc. G represents a monosaccharide, or a straight-chain or branched oligosaccharide containing 2 to 20, more preferably 2 to 12, more preferably 2 to 10, even more preferably 2 to 8, and most preferably 2 to 6 sugar moieties. The sugar moieties that may be present in fragment G are known to those skilled in the art and include, for example, glucose (Glc), galactose (Gal), mannose (Man), trehalose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylglucosamine (GalNAc), N-acetylenolamine (NeuNAc), or sialic acid, xylose (Xyl). The drug moiety (D) of the belentoxidide drug conjugate can be linked to the sugar linker by reacting with functional group A. The drug moiety (D) can be linked to the sugar linker via optional spacers (such as spacers containing, for example, a maleic anhydride moiety). Sugar linkers of these specific embodiments are described, for example, in U.S. Patent Application US 2016 / 0280797.

[0211] In some specific embodiments, the linker is a methylene alkoxyaminocarbamate linker. For methylene alkoxyaminocarbamate linkers, see, for example, RV Kolakowski et al., Angew. Chem. Int. Ed. 2016, 55, 7948-7951 or International Patent Application WO 2015 / 095755, the entire contents of which are incorporated herein by reference.

[0212] In some specific embodiments, the methylene alkoxycarbamate linker has the following structure: , wherein the wavy line indicates the connection with belentoxicin or the antigen-binding fragment (Ab) and the drug portion (D) of the belentoxicin drug conjugate, and the R group is selected from the group consisting of C1-C4 alkyl groups and . Preferably, R is a C1-C4 alkyl group, such as ethyl or . More preferably, R is . In some specific embodiments, the belentoxicin or the antigen-binding fragment (Ab) and the drug portion (D) are connected to the methylene alkoxycarbamate linker as shown in the following structure: , wherein Ab is belentoxicin or the antigen-binding fragment, and D is the drug portion. In these specific embodiments, the R group is selected from the group consisting of C1-C4 alkyl groups and . Preferably, R is a C1-C4 alkyl group, such as ethyl or . More preferably, R is . In some specific embodiments, belenutoxine or the antigen-binding fragment (Ab) and / or the pharmaceutical portion (D) are linked to a methylene alkoxycarbamate linker via an optional spacer. Such a spacer may, for example, have the following structure: , where the wavy line indicates the connection to the methylene alkoxycarbamate linker. This results in the following structure: , where the wavy line indicates a covalent connection of the linker to another portion of the belenutoxine drug conjugate, particularly a covalent connection to belenutoxine or the antigen-binding fragment (Ab) or the pharmaceutical portion (D), preferably a covalent connection to the pharmaceutical portion (D). As a non-limiting example, in a belenutoxine drug conjugate containing such a methylene alkoxycarbamate linker, belenutoxine or its antigen-binding fragment (Ab) may be linked to the maleic anhydride portion, and the pharmaceutical portion (D) may be linked to the position indicated by the wavy line. In these specific embodiments, the R group is selected from the group consisting of C1-C4 alkyl groups and . Preferably, R is a C1-C4 alkyl group, such as ethyl or . Better yet, R is...

[0213] In some specific embodiments, the linker (L) is selected from the following: -(butadieneimin-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(butadieneimin-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(butadieneimin-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(butadieneimin-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2 -C(=O)-, -(butadiimidin-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and -(butadiimidin-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, preferably selected from -(butadiimidin-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-. "D": Drug part

[0214] This document discloses antibody-drug conjugates comprising a pharmaceutical moiety. The terms "pharmaceutical moiety" or "payload" are used interchangeably herein and refer to a chemical or biochemical moiety conjugated with an antibody or antigen-binding fragment. In this regard, the "ADC chemical formula" Ab-(L-(D)x)y described herein is referenced again. Belentuxi can be conjugated with several identical or different pharmaceutical moieties using any method described herein or known in the art. In some preferred embodiments, the pharmaceutical moiety is an anticancer agent. Therefore, the drug can be selected from the group consisting of: camptothecin, topoisomerase inhibitors, maytansinoids, calicheamycin, duocarmycin, tubulolysin, amatoxin, dolastatin, and auristatin, such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimer, indolinobenzodiazepine dimer, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTAC, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogues or prodrugs. In a preferred embodiment, the drug is partially MMAE.

[0215] As used herein, "camptothecin" (CPT) refers to a topoisomerase toxin. It was discovered in 1966 by ME Wall and MC Wani during a systematic screening of natural products for anticancer drugs. It is isolated from the bark and stem of the Camptotheca (Happy tree), native to China and used in traditional Chinese medicine for cancer treatment. The term "camptothecin" may also include CPT analogs. Currently, four CPT analogs are approved for use in cancer chemotherapy: topotecan, irinotecan, belotecan, and trastuzumab deruxtecan. Camptothecin has the following structure:

[0216] CPT term B also includes the following CPT analogues: analog R 1 R 2 R 3 R 4 Topotecan —H —OH —H Irinotecan (CPT-11) —H —H Silatecan (DB-67, AR-67) —H —OH —H Cocinotecan (BNP-1350) —H —H —H Exanotecan —CH3 —F lurtotecan —H Gimatecan (ST1481) —H —H —H Belotticon (CKD-602) —H —H —H Rubitican —H —H —H

[0217] In some specific embodiments, the pharmaceutical portion is a maytansine-like pharmaceutical portion, including those having the following structure: where the wavy line indicates the covalent connection of the sulfur atom of the maytansine-like pharmaceutical portion to the linker of the antibody-drug conjugate. R is independently H or C1-C6 alkyl each time it appears. The alkyl chain that links the amino group to the sulfur atom can be methyl, ethyl, or propyl, i.e., m is 1, 2, or 3. (US Patent No. 633,410, US Patent No. 5,208,020, Chari et al. (1992) Cancer Res. 52:127-131, Lui et al. (1996) Proc. Natl. Acad. Sci. 93:8618-8623).

[0218] The antibody drug conjugates disclosed herein consider all stereoisomers of the maytansine drug moiety, that is, any combination of the R and S configurations on the chiral carbon of maytansine. In some specific embodiments, the maytansine drug moiety has the following stereochemistry:

[0219] In some specific embodiments, the maytansine-like drug portion is N2'-deacetylated-N2'-(3-thiol-1-oxypropyl)-maytansine (also known as DM1). DM1 is represented by the following structure:

[0220] In some specific embodiments, the maytansine-like drug portion is N2'-deacetylated-N2'-(4-thiol-1-oxopentyl)-matansine (also known as DM3). DM3 is represented by the following structure:

[0221] In some specific embodiments, the maytansine-like drug portion is N2'-deacetylated-N2'-(4-methyl-4-thiol-1-oxopentyl)-matansine (also known as DM4). DM4 is represented by the following structure:

[0222] Preferably, in the antibody-drug conjugate containing a maytansine-like drug portion disclosed herein, the maytansine-like drug is N2'-deacetylated-N2'-(3-thiol-1-oxopropyl)-matansine (DM1) or N2'-deacetylated-N2'-(4-methyl-4-thiol-1-oxopentyl)-matansine (DM4).

[0223] The drug component may be chachimycin. As used herein, "chachimycin" refers to a class of enediyne antitumor antibiotics derived from *Micromonospora echinospora*, of which chachimycin γ1 is the most notable. It was initially isolated in the mid-1980s from chalk or "calcareous pits" in Kellville, Texas. It is highly toxic to all cells. Therefore, the drug component may be chachimycin γ1 with the following structural example:

[0224] The drug component may be daunorubicin. As used herein, "daunorubicin" describes a small, synthetic alkylating agent that binds to the minor groove of DNA. Daunorubicin may be suitable for targeting solid tumors. It binds to the minor groove of DNA and alkylates the nucleotide adenine at the N3 position. Irreversible alkylation of DNA disrupts nucleic acid structure, ultimately leading to tumor cell death. Examples of daunorubicin include, but are not limited to, CC-1065, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, and paclitaxel, and their derivatives.

[0225] The drug component may be a platinum-based antitumor agent, such as cisplatin or its derivatives.

[0226] The drug component may be tubulolysin. Tubulolysin possesses anti-microtubule, anti-mitotic, apoptosis-inducing, anti-cancer, anti-angiogenic, and anti-proliferative functions. Tubulolysin is a cytotoxic peptide comprising nine members (AI). Preferably, tubulolysin is tubulolysin A. Tubulolysin A has potential applications as an anticancer agent. It prevents cells from entering the G2 / M phase. Tubulolysin A has the following structure:

[0227] The drug component may be amatoxins. Amatoxins are a collective term for at least eight related toxic compounds found in several genera of poisonous mushrooms, most notably *Amanita phalloides* and several other members of the *Amanita* genus, as well as some species of mushrooms in the genera *Conocybe*, *Galerina*, and *Lepiota*. Even small doses of amatoxins can be fatal. These compounds have similar structures, with eight amino acid residues arranged in a conserved large bicyclic motif (which is a pentcyclic structure overall when considering the inherent rings in proline and tryptophan-derived residues). All amatoxins are oligopeptides synthesized as a preprotein of 35 amino acids, the last eight of which are cleaved by prolyl oligopeptidase. The schematic amino acid sequence of amatoxins is Ile-Trp-Gly-Ile-Gly-Cys-Asn-Pro, where Trp and Cys are partially cross-linked via a monoxide (S=O) moiety and hydroxylated in molecular variants. Currently, ten amatoxins are known to be potential pharmaceutical components: name R 1 R 2 R 3 R 4 R 5 α-Amanitin OH OH NH2 OH OH β-Amanitain OH OH OH OH OH γ-Amanitain OH H NH2 OH OH ε-Amanitain OH H OH OH OH Amanullin, a non-toxic cyclic peptide. H H NH2 OH OH Amanullinic acid (hydroxyamanitin carboxylic acid) H H OH OH OH Amaninamide OH OH NH2 H OH Amanin OH OH OH H OH Proamanulin H H NH2 OH H

[0228] The drug component may be telsonin, such as telsonin 10 or telsonin 15. Both are marine natural products isolated from the Indian Ocean sea hare *Dollabella auricularia*. This potent antitumor agent has also been isolated from the marine cyanobacterium *Symploca cyanobacterium* sp. VP642 in Palau. As small linear peptide molecules, telsonin 10 and 15 are considered anticancer drugs, showing efficacy against breast and liver cancer, solid tumors, and some leukemias. Preclinical studies have demonstrated efficacy in experimental antitumor and microtubule assembly systems. Telsonin is a mitotic inhibitor. It inhibits microtubule assembly by interfering with microtubule formation, thereby disrupting cell division through mitosis and inducing apoptosis and Bcl-2 phosphorylation in several malignant cell types. Tail harein 10 (N,N-dimethyl-L-valine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazolyl-2-yl)ethyl]amino}propyl]-1-pyrrolidinyl}-5-methyl-1-oxo-4-heptyl]-N-methyl-L-valine) has the following structure:

[0229] Tail slugin 15 ((2S)-1-[(2S)-2-benzyl-3-methoxy-5-oxo-2,5-dihydro-1H-pyrrole-1-yl]-3-methyl-1-oxo-2-butylN,N-dimethyl-L-valine-L-valine-N-methyl-L-valine-L-proline-L-proline) has the following structure:

[0230] In some specific embodiments of the antibody-drug conjugate disclosed herein, the drug portion is aprestatin. Preferably, aprestatin is monomethylaprestatin F (MMAF) or monomethylaprestatin E (MMAE).

[0231] In some specific embodiments of the antibody-drug conjugate described herein, the drug portion is monomethylarestatin F (also known as MMAF). MMAF is represented by the following structural formula: MMAF. Monomethylarestatin F (MMAF) can be bound to a linker via a nitrogen atom marked with an asterisk (*).

[0232] In some specific embodiments, the aprestatin pharmaceutical portion is monomethylaprestatin E (also known as MMAE). MMAE is represented by the following structural formula: MMAE. Monomethylaprestatin E (MMAE) can be linked to a linker via a nitrogen atom marked with an asterisk (*).

[0233] These molecules noncompetitively inhibit the binding of vincristine to tubulin (at a location known as the vinca / peptide region), but have been shown to bind to the RZX / MAY region.

[0234] The pharmaceutical part may be a pyrrolobenzodiazepine dimer, such as a compound having the following structure:

[0235] The pharmaceutical part may be an indoline benzodiazepine dimer, such as a compound having the following structure:

[0236] The pharmaceutical component may be a radioactive isotope. Typical radioactive isotopes as described herein may involve small radiation sources, typically gamma or beta emitters, such as iodine-125, iodine-131, iridium-192, or palladium-103.

[0237] The drug component may be a therapeutic protein or peptide or fragment thereof. Typical examples are cytokines such as interleukin, ricin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin PE38.

[0238] The drug component may be a kinase inhibitor, preferably a kinase inhibitor associated with tumorigenesis. Exemplary kinase inhibitors include imatinib, nilotinib, dasatinib, bosutinib, ponatinib, gefitinib, erlotinib, afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, pazopanib, axitinib, lenvatinib, cabozantinib, vandetanib, and regorafenib. (ib), vemurafenib, dabrafenib, trametinib, cobimetinib, crizotinib, certinib, alectinib, brigatinib, lorlatinib, ibrutinib, acalibrutinib, midostaurin, ruxolitinib, idelalisib, copanlisib, palbociclib, ribociclib, or abemaciclib.

[0239] The drug component may be a MEK inhibitor. As described herein, a MEK inhibitor is a chemical or drug that inhibits mitogen-activated protein kinases MEK1 and / or MEK2. It can be used to affect the MAPK / ERK pathway, which is typically overactive in some cancers. Therefore, MEK inhibitors have the potential to treat certain cancers, particularly BRAF-mutant melanoma and KRAS / BRAF-mutant colorectal cancer. Typical MEK inhibitors include trametinib (GSK1120212), cobimetinib or XL518, binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901, or TAK-733.

[0240] The drug component may be a KSP (kinesin spindle) inhibitor. Examples of KSP inhibitors include ispinesib (SB-715992), SB743921, AZ 3146, GSK923295, BAY 1217389, MPI-0479605, and ARQ 621.

[0241] The drug component may be a nucleic acid. When used as a drug component, the nucleic acid may involve DNA / RNA molecules, such as DNA / RNA molecules with immunomodulatory functions (preferably for innate immunity). Immunomodulation may involve increasing or decreasing the immune response, preferably decreasing it. The DNA / RNA molecule may also be siRNA, preferably designed for (specifically) regulating / manipulating (e.g., reducing) the expression of a target protein (an exemplary target may be detrophic myotonic kinase (DMPK)).

[0242] The drug component may be a PROTAC. A target proteolytic chimera (PROTAC) is a heterobifunctional small molecule containing two active domains and a linker capable of removing a specific unwanted protein. PROTACs do not function as typical enzyme inhibitors, but rather exert their effects by inducing selective intracellular proteolysis. A PROTAC generally comprises two covalently linked protein-binding molecules: one that binds to an E3 ubiquitin ligase, and the other that binds to the target protein to be degraded. The E3 ligase attracts the target protein, leading to ubiquitination, and the target protein is subsequently degraded by the proteasome. An exemplary PROTACS is described in Sakamoto et al. (2001), PNAS, 98(15):8554–9, which is incorporated herein by reference. Methods for manufacturing ADCs

[0243] The method for manufacturing the ADC of the present invention is known in the art. Specifically, WO 2016 / 066749, WO 2017 / 186855, Schumacher et al., Angew. Chem. Int. Ed. 2015, 54, 13787-13791 have described the introduction or addition of a microtubule-tyrosine ligase recognition sequence at the C-terminus of a polypeptide, contacting the polypeptide (here, belentoxicet) with a non-natural amino acid in the presence of microtubule-tyrosine ligase, and conjugating a linker containing the drug moiety with the linked belentoxicet. All of the above-mentioned documents are incorporated herein by reference. The Materials and Methods section of the examples also contains instructions on how to manufacture or obtain the ADC of the present invention.

[0244] Therefore, the present invention further relates to a method of manufacturing an ADC as defined herein, comprising: (a) introducing or adding a microtubule tyrosine ligase recognition sequence to the C-terminus of a light chain, a heavy chain, or both a light chain and a heavy chain of belentoxidase; (b) contacting the belentoxidase obtained in step (a) in the presence of microtubule tyrosine ligase and a non-natural amino acid, under conditions suitable for microtubule tyrosine ligase to link the belentoxidase to the non-natural amino acid; and (c) conjugating an optional cleavable linker comprising a pharmaceutical moiety to the linked belentoxidase obtained in step (b).

[0245] As described herein, a TTL recognition sequence is introduced or added to the C-terminus of belentoxigenic acid. For example, this recognition sequence may be introduced or added by genetic engineering or by synthesis (chemical protein synthesis or via synthetic biology).

[0246] The present invention further relates to an ADC obtainable by a method of manufacturing an ADC as defined herein. The present invention further relates to an ADC obtainable by a method of manufacturing an ADC as defined herein. Pharmaceutical Composition

[0247] The present invention further relates to pharmaceutical compositions comprising the ADC of the present invention. Pharmaceutical compositions according to the present invention may further comprise one or more pharmaceutically acceptable carriers. In a particular specific embodiment, the term "pharmaceutically acceptable" refers to approval by a regulatory authority or other recognized pharmacopoeia for use in animals, and more specifically for use in humans. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water, 5% glucose or physiologically buffered saline, or other solvents or mediators such as ethylene glycol, glycerol, oils such as olive oil, or injectable organic esters suitable for administration to human or non-human individuals. Specific exemplary pharmaceutically acceptable carriers include (biodegradable) liposomes; microspheres made of the biodegradable polymer poly(D,L-lactic acid-co-glycolic acid) (PLGA), albumin microspheres; synthetic polymers (soluble); nanofibers, protein-DNA complexes; protein conjugates; red blood cells; or viruses. Various carrier-based dosage forms include the following: solid lipid nanoparticles (SLN), polymer nanoparticles, ceramic nanoparticles, hydrogel nanoparticles, copolymer peptide nanoparticles, nanocrystals and nanosuspensions, nanocrystals, nanotubes and nanowires, functionalized nanocarriers, nanospheres, nanocapsules, liposomes, lipid emulsions, lipid microtubules / microcylinders, lipid microvesicles, lipoglobules, lipopolyplexes, antilipid microcells, dendritic polymers, ethosomes, multicomponent ultrathin capsules, aquasomes, pharmacosomes, colloidosomes, niosomes, discoids, proniosomes, microspheres, microemulsions, and polymer microcells. Other suitable pharmaceutically acceptable carriers and excipients are specifically described in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey (1991). See also, for example, Remington: The Science and Practice of Pharmacy, 21st edition; Lippincott Williams & Wilkins, 2005.

[0248] In some specific embodiments, the pharmaceutically acceptable carrier or composition is sterile. In addition to active agents, the pharmaceutical composition may also contain physiologically acceptable compounds, such as fillers, additives, solubilizers, stabilizers, permeabilizers, absorption enhancers, etc. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose, lactose; dextran; polyols such as mannitol; antioxidants such as ascorbic acid or glutamic acid thiosulfate; preservatives; chelating agents; buffers; or other stabilizers or excipients.

[0249] The choice of pharmaceutically acceptable carriers and / or physiologically acceptable compounds may depend on, for example, the nature of the active agent, such as solubility, compatibility (meaning that these substances can coexist in the composition without interacting with each other in a way that would significantly reduce the efficacy of the pharmaceutical composition under normal use) and / or the route of administration of the composition.

[0250] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the ADC described herein and can be formulated in various forms, such as solid, liquid, gas, or lyophilized forms, and can be particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, inhalers, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or liquid extracts, or in forms particularly suitable for topical or oral administration. Multiple routes are suitable for the administration of the polypeptides of the present invention, including but not limited to oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, or intraocular administration. However, any other route can be readily chosen by those skilled in the art if desired. Use in therapeutic applications

[0251] As shown in Examples 4 and 5, the ADC of the present invention can be used for treatment, particularly for the treatment of cancer. Therefore, the present invention further relates to the use of the ADC of the present invention or the pharmaceutical composition of the present invention in a method of treating a disease, optionally comprising administering an effective amount of the ADC of the present invention or the pharmaceutical composition of the present invention to an individual or patient in need of it. Preferably, the disease is related to the overexpression of CD30. The disease may be cancer related to the overexpression of CD30. More preferably, the diseases are selected from the following groups: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and bowel lesion-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, especially systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). Diseases can be selected from the following groups: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. Diseases can be peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL). Diseases can be cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). Diseases can be Hodgkin's lymphoma (HL).

[0252] This invention also relates to the use of the ADC of the present invention in the preparation of a medicament for treating a disease. This invention also relates to the use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease. Preferably, the disease is related to the overexpression of CD30. The disease may be a cancer related to the overexpression of CD30. More preferably, the disease is selected from the group consisting of: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell cancer; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). Diseases can be selected from the following groups: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. Diseases can be peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL). Diseases can be cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). Diseases can be Hodgkin's lymphoma (HL).

[0253] This invention also relates to a method of treating a disease, comprising administering an effective amount of the ADC of the present invention to an individual or patient in need of it. Preferably, the disease is related to the overexpression of CD30. The disease may be cancer related to the overexpression of CD30. More preferably, the diseases are selected from the following groups: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and bowel lesion-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, especially systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). The disease may include, but is not limited to, the following: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, or follicular T-cell lymphoma. The disease may also be peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL) or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). The disease may also be Hodgkin's lymphoma (HL).

[0254] The term "effective amount" refers to the amount of a therapeutic agent (e.g., the ADC of the present invention) that, when used alone or in combination with another therapeutic agent, protects an individual against the onset of disease or promotes disease remission, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of injury or disability caused by disease-related suffering. The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to those skilled in the art, such as in human individuals during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in an in vitro assay. The exact amount will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0255] Additionally, the present invention relates to antibody-drug conjugates as disclosed herein, used in methods of treating cancer in patients. According to the invention, the term "patient" refers to a human, a non-human primate, or another animal, particularly a mammal such as a cow, horse, pig, sheep, goat, dog, cat, or rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human. Unless otherwise stated, the terms "patient" or "individual" are used interchangeably herein. The term "treatment" in all its grammatical forms includes therapeutic or preventative treatment. "Therapeutic or preventative treatment" includes preventative treatment aimed at completely preventing clinical and / or pathological manifestations, or therapeutic treatment aimed at improving or alleviating clinical and / or pathological manifestations. Therefore, the term "treatment" also includes the improvement or prevention of disease.

[0256] The ADC of the present invention can be administered at any therapeutically effective dose. The upper limit generally refers to a dose that is still safe to administer with respect to side effects. Typically, the ADC of the present invention can be administered at an effective dose of 0.5 to 20 mg / kg. The ADC of the present invention can be administered at an effective dose of 1 to 10 mg / kg. The ADC of the present invention can be administered at an effective dose of 1 to 9 mg / kg. The ADC of the present invention can be administered at an effective dose of 1.2 to 9 mg / kg. The ADC of the present invention can be administered at an effective dose of 1.8 to 8 mg / kg. The ADC of the present invention can be administered at an effective dose of 2 to 6 mg / kg. As illustrative examples, the ADC of the present invention can be administered at effective doses of 20 mg / kg, 18 mg / kg, 16 mg / kg, 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. Other dosages may be used if deemed beneficial or necessary.

[0257] The ADC of the present invention can be co-administered with other agents, particularly anticancer drugs, or compounds that enhance the effects of these drugs. Co-administration includes sequential administration and simultaneous administration. Suitable anticancer drugs include, for example, one or more of the drug components described herein. The ADC of the present invention can be co-administered with cyclophosphamide, doxorubicin, and prednisone (CHP regimen). The ADC of the present invention can be co-administered with anticancer drugs, such as immunomodulatory drugs, checkpoint inhibitors, chemotherapeutic agents (e.g., cyclophosphamide, doxorubicin hydrochloride (hydroxydanomycin), vincristine sulfate (Oncovin), and prednisone (CHOP regimen), etoposide, vincristine, nitrogen mustard), protein inhibitors, etc.

[0258] "Tumor" refers to a group of cells or tissues formed by disordered cell proliferation, particularly cancer. Tumors may exhibit partial or complete lack of structural tissue and functional coordination with normal tissue, and typically form a distinctive tissue mass, which can be benign or malignant. In particular, the term "tumor" refers to malignant tumors. According to one specific embodiment, the terms "tumor" or "tumor cells" also refer to non-solid cancers and cells of non-solid cancers, such as leukemia cells. According to another specific embodiment, the terms "tumor" and "tumor cells" do not cover the corresponding non-solid cancers or their cells.

[0259] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and typically involves cancer cells detaching from the primary tumor, entering the circulation, and settling to grow in normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are usually similar to those in the original tumor. This means, for example, if breast cancer metastasizes to the lungs, the secondary tumor will be composed of abnormal breast cells, not abnormal lung cells. The tumor in the lungs is called metastatic breast cancer, not lung cancer. Exemplary ADC of the present invention

[0260] An overview of exemplary ADCs is disclosed in the table below. In those exemplary ADCs, the non-natural amino acid is 3-methoxytyrosine, and the pharmaceutical part (D) is a pharmaceutical part as defined herein, preferably MMAE: Berentoxi light chain Berentoxice Heavy Chain connector SEQ ID NO: 2 SEQ ID NO: 12 (Includes TTL identification sequence) Structure 4 SEQ ID NO: 2 SEQ ID NO: 12 Structure 5 SEQ ID NO: 11 (Includes TTL identification sequence) SEQ ID NO: 1 Structure 4 SEQ ID NO: 11 SEQ ID NO: 1 Structure 5 SEQ ID NO: 11 SEQ ID NO:12 Structure 4 SEQ ID NO: 11 SEQ ID NO: 12 Structure 5 SEQ ID NO: 14 (Includes TTL identification sequence) SEQ ID NO: 1 Structure 4 SEQ ID NO: 14 SEQ ID NO: 1 Structure 5 SEQ ID NO: 14 SEQ ID NO: 12 Structure 4 SEQ ID NO: 14 SEQ ID NO: 12 Structure 5

[0261] Structures 4 and 5 are repeated as follows:

[0262] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

[0263] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each heavy chain; each heavy chain, including the recognition sequence, has SEQ ID NO: 12; and each light chain of belentoxigenin has SEQ ID NO: 2; and (b) the C-terminus of the recognition sequence of each heavy chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each heavy chain.

[0264] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each heavy chain; each heavy chain, including the recognition sequence, having SEQ ID NO: 12; and wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, having SEQ ID NO: 11; and (b) the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain are bound via amide bonds to groups having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain.

[0265] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

[0266] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 14; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

[0267] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each heavy chain; each heavy chain, including the recognition sequence, having SEQ ID NO: 12; and wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, having SEQ ID NO: 14; and (b) the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain are bound via amide bonds to groups having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain.

[0268] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin comprises a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 14; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain. Sequence

[0269] This article reveals that the following sequences are particularly relevant.

[0271] Belantamab / cAC10 light chain (SEQ ID NO: 2): Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Val-Ser-Leu-Gly-Gln-Arg-Ala-Thr-Ile-Ser-Cys-Lys-Ala-Ser-Gln-Ser-Val-Asp-Phe-Asp-Gly-Asp-Ser-Tyr-Met-Asn-Trp-Tyr-Gln-Gln-Lys-Pro-Gly-Gln-Pro-Pro-Lys-Val-Leu-Ile-Tyr-Ala-Ala-Ser-Asn-Leu-Glu-Ser-Gly-Ile-Pro-Ala-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Asp-Phe-Thr-Leu-Asn-Ile-His-Pro-Val-Glu-Glu-Glu-Asp-Ala-Ala-Thr-Tyr-Tyr-Cys-Gln-Gln-Ser-Asn-Glu-Asp-Pro-Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-Arg-Thr-Val-Ala-Ala-Pro-Ser-Val-Phe-Ile-Phe-Pro-Pro-Ser-Asp-Glu-Gln-Leu-Lys-Ser-Gly-Thr-Ala-Ser-Val-Val-Cys-Leu-Leu-Asn-Asn-Phe-Tyr-Pro-Arg-Glu-Ala-Lys-Val-Gln-Trp-Lys-Val-Asp-Asn-Ala-Leu-Gln-Ser-Gly-Asn-Ser-Gln-Glu-Ser-Val-Thr-Glu-Gln-Asp-Ser-Lys-Asp-Ser-Thr-Tyr-Ser-Leu-Ser-Ser-Thr-Leu-Thr-Leu-Ser-Lys-Ala-Asp-Tyr-Glu-Lys-His-Lys-Val-Tyr-Ala-Cys-Glu-Val-Thr-His-Gln-Gly-Leu-Ser-Ser-Pro-Val-Thr-Lys-Ser-Phe-Asn-Arg-Gly-Glu-Cys

[0272] TTL recognition sequence (SEQ ID NO: 3): Xaa1-Xaa2-Xaa3-Glu, wherein: Xaa1 is any amino acid; as an illustrative example, Xaa1 may be Glu, Asp, Ala, Lys or Pro; Xaa2 is any amino acid; as an illustrative example, Xaa2 may be Gly, Ser, Ala, Val or Phe; and Xaa3 is Glu, Asp or Cys.

[0273] TTL identification sequence (SEQ ID NO: 4): Glu-Gly-Glu-Glu

[0274] TTL recognition sequence (SEQ ID NO: 5): Val-Asp-Ser-Val-Glu-Gly-Glu-Gly-Glu-Glu-Glu-Gly-Glu-Glu

[0275] TTL recognition sequence (SEQ ID NO: 6): Ser-Val-Glu-Gly-Glu-Gly-Glu-Glu-Glu-Gly-Glu-Glu

[0276] TTL identification sequence (SEQ ID NO: 7): Ser-Ala-Asp-Gly-Glu-Asp-Glu-Gly-Glu-Glu

[0277] TTL identification sequence (SEQ ID NO: 8): Ser-Val-Glu-Ala-Glu-Ala-Glu-Glu-Gly-Glu-Glu

[0278] TTL identification sequence (SEQ ID NO: 9): Ser-Tyr-Glu-Asp-Glu-Asp-Glu-Gly-Glu-Glu

[0279] TTL identification sequence (SEQ ID NO: 10): Ser-Phe-Glu-Glu-Glu-Asn-Glu-Gly-Glu-Glu

[0280] Berentoxicin light chain, comprising a Gly-Gly-Gly-Gly-Ser- linker and a TTL recognition sequence at the C-terminus (SEQ ID NO: 11):Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Val-Ser-Leu-Gly-Gln-Arg-Ala-Thr-Ile-Ser-Cys-Lys-Ala-Ser-Gln-Ser-Val-Asp-Phe-Asp-Gly-Asp-Ser-Tyr-Met-Asn-Trp-Tyr-Gln-Gln-Lys-Pro-Gly-Gln-Pro-Pro-Lys-Val-Leu-Ile-Tyr-Ala-Ala-Ser-Asn-Leu-Glu-Ser-Gly-Ile-Pro-Ala-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Asp-Phe-Thr-Leu-Asn-Ile-His-Pro-Val-Glu-Glu-Glu-Asp-Ala-Ala-Thr-Tyr-Tyr-Cys-Gln-Gln-Ser-Asn-Glu-Asp-Pro-Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-Arg-Thr-Val-Ala-Ala-Pro-Ser-Val-Phe-Ile-Phe-Pro-Pro-Ser-Asp-Glu-Gln-Leu-Lys-Ser-Gly-Thr-Ala-Ser-Val-Val-Cys-Leu-Leu-Asn-Asn-Phe-Tyr-Pro-Arg-Glu-Ala-Lys-Val-Gln-Trp-Lys-Val-Asp-Asn-Ala-Leu-Gln-Ser-Gly-Asn-Ser-Gln-Glu-Ser-Val-Thr-Glu-Gln-Asp-Ser-Lys-Asp-Ser-Thr-Tyr-Ser-Leu-Ser-Ser-Thr-Leu-Thr-Leu-Ser-Lys-Ala-Asp-Tyr-Glu-Lys-His-Lys-Val-Tyr-Ala-Cys-Glu-Val-Thr-His-Gln-Gly-Leu-Ser-Ser-Pro-Val-Thr-Lys-Ser-Phe-Asn-Arg-Gly-Glu-Cys-Gly-Gly-Gly-Gly-Ser-Val-Asp-Ser-Val-Glu-Gly-Glu-Gly-Glu-Glu-Glu-Gly-Glu-Glu

[0283] Belentoxicin light chain containing a TTL recognition sequence (SEQ ID NO: 14) at the C-terminus: Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Val-Ser-Leu-Gly-Gln-Arg-Ala-Thr-Ile-Ser-Cys-Lys-Ala-Ser-Gln-Ser-Val-Asp-Phe-Asp-Gly-Asp-Ser-Tyr-Met-Asn-Trp-Tyr-Gln-Gln-Lys-Pro-Gly-Gln-Pro-Pro-Lys-Leu-Ile-Tyr-Ala-Ala-Ser-Asn-Leu -Glu-Ser-Gly-Ile-Pro-Ala-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Asp-Phe-Thr-Leu-Asn-Ile-His-Pro-Val-Glu-Glu-Glu-Asp-Ala -Ala-Thr-Tyr-Tyr-Cys-Gln-Gln-Ser-Asn-Glu-Asp-Pro-Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-Arg-Thr-Val-Ala-Ala -Pro-Ser-Val-Phe-Ile-Phe-Pro-Pro-Ser-Asp-Glu-Gln-Leu-Lys-Ser-Gly-Thr-Ala-Ser-Val-Val-Cys-Leu-Leu-Asn-Asn-Phe-Tyr-Pro -Arg-Glu-Ala-Lys-Val-Gln-Trp-Lys-Val-Asp-Asn-Ala-Leu-Gln-Ser-Gly-Asn-Ser-Gln-Glu-Ser-Val-Thr-Glu-Gln-Asp-Ser-Lys-Asp -Ser-Thr-Tyr-Ser-Leu-Ser-Ser-Thr-Leu-Thr-Leu-Ser-Lys-Ala-Asp-Tyr-Glu-Lys-His-Lys-Val-Tyr-Ala-Cys-Glu-Val-Thr-His-Gln-Gly-Leu-Ser-Ser-Pro-Val-Thr-Lys-Ser-Phe-Asn-Arg-Gly-Glu-Cys-Val-Asp-Ser-Val-Glu-Gly ...An antibody-drug conjugate (ADC) comprising: (a) brentuximab, wherein the brentuximab comprises a microtubule tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, heavy chain, or all of the heavy chain and the light chain; and (b) at least one pharmaceutical moiety; wherein the pharmaceutical moiety is conjugated to each of the non-natural amino acids via a linker. 2. The ADC of claim 1, wherein the heavy chain of brentuximab has an amino acid sequence comprising SEQ ID NO: 1 or having at least 95% sequence identity with SEQ ID NO: 1, and / or wherein the light chain of brentuximab has an amino acid sequence comprising SEQ ID NO: 2 or having at least 95% sequence identity with SEQ ID NO: 2; preferably, brentuximab comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 1, and the light chain comprises the amino acid sequence of SEQ ID NO: 2. 3. The ADC of any of the foregoing items, wherein the drug portion is selected from the group consisting of: camptothecin, maytansinoid, calicheamycin, duocarmycin, tubulolysin, amatoxin, dolastatin, and auristatin, such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimer, indolinobenzodiazepine dimer, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTAC, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogues or prodrugs thereof; preferably, the drug portion is MMAE. 4. The ADC of any of the preceding items, wherein the recognition sequence of the tubulin tyrosine ligase has at least the amino acid sequence X1X2X3X4 (SEQ ID NO: 3), wherein X1 and X2 are any amino acids, X3 is E, D or C, and X4 is E; preferably, X2 is G, S, A, V or F, and / or X1 is E, D, A, K or P. 5. The ADC of any of the preceding items, wherein the recognition sequence is EGEE (SEQ ID No. 4), preferably VDSVEGEGEEEGEE (SEQ ID No. 5), SVEGEGEEEGEE (SEQ ID No. 6), SADGEDEGEE (SEQ ID No. 7), SVEAEAEEGEE (SEQ ID No. 8), SYEDEDEGEE (SEQ ID No. 9), or SFEEENEGEE (SEQ ID No. 10). 6.7. An ADC of any of the preceding items, wherein the non-natural amino acid is a 2-substituted, 3-substituted, or 4-substituted tyrosine, or a tyrosine derivative substituted at the benzyl position. 8. An ADC of item 6, wherein the 3-substituted or 4-substituted tyrosine derivative is 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-methoxytyrosine, 3-acetyrosine, or 4-aminophenylalanine; preferably, the non-natural amino acid is 3-methoxytyrosine. 9. An ADC of any of the preceding items, wherein the linker is cleavable, preferably cleavable by a protease, and more preferably cleavable by a cathepsin (such as cathepsin B). 10. An ADC of any of items 1 to 7, wherein the linker is non-cleavable. 11. An ADC of any of the preceding items, wherein the linker comprises a valine-citrulline moiety. 10. An ADC of any of the preceding claims, wherein the linker comprises a hydroxylamine group and the non-natural amino acid comprises a formyl group located ortho to a hydroxyl group in an aromatic ring (such as 3-methoxytyrosine), and wherein, after conjugation, the hydroxylamine group of the linker forms an oxime with the formyl group of the non-natural amino acid. 11. An ADC of any of the preceding claims, wherein belentoxicin is conjugated with two, four, six, or eight, preferably two or four, and more preferably two, the drug moieties. 12. An ADC of any of the preceding claims, wherein the linker has a structure as shown in Structure 1 before conjugation with the non-natural amino acid: where R is one or more drug moieties, optionally conjugated with the hydroxylamine of Structure 1 via one or more cleavage sites, preferably wherein the hydroxylamine of Structure 1 is conjugated with the non-natural amino acid. 13. An ADC of any of the foregoing items, wherein the linker has a structure as shown in structure 2 or 3 before conjugation with the non-natural amino acid: wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; wherein D is one or more pharmaceutical moieties; and wherein Y is a cleavage site, such as a cleavage site of a cathepsin (such as cathepsin B); preferably wherein the hydroxylamine of structure 2 or 3 is conjugated with the non-natural amino acid. 14. An ADC as described in any of the preceding items, wherein the linker has a structure as shown in structure 4 or 5 before conjugation with the non-natural amino acid, wherein D is the pharmaceutical moiety, preferably MMAE. 15. An ADC as described in item 14, wherein D is MMAE, wherein the non-natural amino acid is 3-methoxytyrosine, and the hydroxylamine group of the linker forms an oxime with the 3-methoxy group of the non-natural amino acid. 16.An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein each light chain of belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at its C-terminus; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain. 17. An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each heavy chain; each heavy chain, including the recognition sequence, has SEQ ID NO: 12; and each light chain of belentoxigenin has SEQ ID NO: 2; and (b) the C-terminus of the recognition sequence of each heavy chain is bound via an amide bond to a group having the following structure: wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each heavy chain. 18. An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each heavy chain; each heavy chain, including the recognition sequence, having SEQ ID NO: 12; and wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, having SEQ ID NO: 11; and (b) the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain are bound via amide bonds to groups having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each heavy chain and the C-terminus of the recognition sequence of each light chain. 19. An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein each light chain of belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at its C-terminus; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1; and (b) the C-terminus of the recognition sequence of each light chain is bound via a amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain. 20.A method of manufacturing an ADC as defined in any one of items 1 to 19, comprising: (a) introducing or adding a microtubule-tyrosine ligase recognition sequence to the C-terminus of the light chain, heavy chain, or both the light and heavy chains of belentoxicin; (b) contacting the belentoxicin obtained in step (a) in the presence of microtubule-tyrosine ligase and a non-natural amino acid, under conditions suitable for microtubule-tyrosine ligase to link the belentoxicin to the non-natural amino acid; and (c) conjugating an optional cleavable linker comprising a pharmaceutical moiety to the linked belentoxicin obtained in step (b). 21. An ADC that may be obtained or is being obtained by the method of item 20. 22. A pharmaceutical composition comprising an ADC of any one of items 1 to 19 or 21. 23. A method of using an ADC of any one of items 1 to 19 or 21 or a pharmaceutical composition of item 22 for the treatment of a disease. 24. The ADC or pharmaceutical composition for use as described in item 23, wherein the disease is associated with overexpression of CD30. 25. The use of the ADC or pharmaceutical composition for use as described in item 23 or 24, wherein the disease is selected from the group consisting of: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 25a. Use of the ADC or pharmaceutical composition for use as described in item 23 or 24, wherein the disease is selected from the group consisting of: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 25b. Use of the ADC or pharmaceutical composition for use as described in item 23 or 24, wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). 26. The use of an ADC as described in items 1 to 19 or 21, for the manufacture of a medicament for treating diseases associated with overexpression of CD30. 27.As per the use of item 26, the disease is selected from the following groups: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE). 28. As per the use of item 27, the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 28a. As used in item 26, wherein the disease is selected from the group consisting of: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 28b. As used in item 26, wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). 29. Use of a pharmaceutical composition as described in item 22 for manufacturing a medicament for treating diseases associated with CD30 overexpression. 30. As used in item 29, wherein the disease is selected from the group consisting of: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE). 31. As used in item 30, wherein the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 31a.As used in item 29, wherein the disease is selected from the group consisting of: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 31b. As used in item 29, wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). 32. A method of treating a disease associated with CD30 overexpression, comprising administering an effective amount of an ADC as described in any one of items 1 to 19 to an individual or patient in need of it. 33. The method of item 32, wherein the disease is a cancer associated with CD30 overexpression. 34. The method of item 32 or 33, wherein the disease is selected from the group consisting of: lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 35. The method of item 34, wherein the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 35a. The method of item 32 or 33, wherein the disease is selected from the group consisting of: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 35b. The method of item 32 or 33, wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). 36.The method of any one of items 32 to 35b, wherein the ADC is administered at a dose of 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg; or at a dose of 2–6 mg / kg. 37. A method for treating a disease associated with CD30 overexpression, comprising administering an effective amount of a pharmaceutical composition of item 22 to an individual or patient in need of it. 38. The method of item 37, wherein the disease is cancer associated with CD30 overexpression. 39. The method of item 37 or 38, wherein the disease is selected from the group consisting of: lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE); preferably Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 40. The method of item 39, wherein the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL). 40a. The method of item 37 or 38, wherein the disease is selected from the group consisting of: nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 40b. The method of item 37 or 38, wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL). 41. The method of any of items 37 to 40b, wherein the ADC is administered at a dose of 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg; or at a dose of 2–6 mg / kg. ****.

[0284] Please note that, as used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “reagent” includes one or more such different reagents, and reference to “method” includes reference to equivalent steps and methods known to those skilled in the art, which may be modified or substituted for the methods described herein.

[0285] Unless otherwise stated, the word "at least" preceding a series of elements should be understood to refer to each element in that series. Those skilled in the art will recognize that many equivalent embodiments of the invention described herein can be determined, or may not require the use of typical experiments. Such equivalent embodiments are intended to be covered in this invention.

[0286] The word “and / or” as used herein includes the meanings of “and”, “or” and “all or any other combination of elements connected by the term”.

[0287] Words such as "less than" or "greater than" do not include specific numbers.

[0288] For example, less than 20 means less than the indicated number. Similarly, more than or greater than means more than or greater than the indicated number, such as more than 80% involving more than or greater than 80% of the indicated number.

[0289] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprising” and variations thereof, such as “comprising” and “including”, shall be construed as implying the inclusion of the stated integers or steps or groups of integers or steps, but not excluding any other integers or steps or groups of integers or steps. When used herein, the word “comprising” may be replaced by words such as “containing” or “including”, or sometimes by the word “having” when used herein. When used herein, “consisting of” excludes any unspecified elements, steps, or components.

[0290] The word "including" includes "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0291] As used herein, the terms “about,” “approximately,” or “substantially” mean within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. This also includes specific numbers, that is, “about 20” includes the number 20.

[0292] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents, and substances described herein, and therefore may vary. The terminology used herein is for the purpose of describing specific particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0293] All references cited in this specification (including all patents, patent applications, scientific literature, specifications, etc.), whether above or below, are incorporated herein by reference in their entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to such disclosure based on prior invention. To the extent that any material incorporated by reference contradicts or is inconsistent with this specification, this specification shall supersede any such material.

[0294] All references and patent documents cited herein are incorporated herein in their entirety. Examples

[0295] The invention and its advantages will be better understood from the following examples, which are for illustrative purposes only. These examples are not intended to limit the scope of the invention in any way. Materials and Methods: Chemicals, Materials, Reagents, and Solvents

[0296] Chemicals and solvents were purchased from Merck (Merck group, Germany), TCI (Tokyo Chemical Industry CO., LTD., Japan), Acros Organics (Thermo Fisher Scientific, USA), Sigma-Aldrich (Merck group, Germany), Carl Roth (Carl Roth GmbH, Germany), and Cytiva (Cytiva UK Limited, UK), and were ready for use without further purification. Anhydrous solvents were purchased from Acros Organics (Thermo Fisher Scientific, USA). TTL performance and purification.

[0297] As previously described (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791, WO 2016 / 066749, WO 2017 / 186855), tubulin-tyrosine ligase (TTL) was expressed in *Escherichia coli* BL21(DE3) with N-terminal His- and SUMO3 soluble tags. Cells were cultured in LB medium supplemented with kanamycin (30 µg / mL) at 37°C and 180 rpm until an OD600 of 0.6 to 0.8 was achieved. After cooling the culture to 18°C, cells were induced with 0.5 mM IPTG, and protein expression was completed at 18°C ​​for 18 hours.

[0298] After collecting the culture by centrifugation, it was lysed by sonic oscillation in TTL binding buffer (20 mM Tris / HCl pH 8.2, 250 mM NaCl, 20 mM imidazole, 3 mM β-thiol ethanol) supplemented with lysozyme (100 µg / ml), DNase (25 µg / ml) and PMSF (1 mM). The fragments were then centrifuged at 20,000 g for 30 minutes and the supernatant was filtered.

[0299] His-SUMO3-TTL was purified using a linear gradient exceeding 4 CV with 5 ml His-Trap HP (Cytiva) to flushing buffer (20 mM Tris / HCl pH 8.2, 250 mM NaCl, 500 mM imidazole, 3 mM β-thiol ethanol). The purified protein was then desalted on a PD-10 Sephadex G-25M column (Cytiva) by buffer exchange to TTL storage buffer (20 mM MOPS / KCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 50 mM L-arginine, 3 mM β-thiol ethanol). Protein aliquots were rapidly frozen and stored at -80°C. Belentoxicin Tub-tag performance and purification.

[0300] Recombinant expression of belentoxigenic Tub-tag was performed in suspension-adapted CHO cells. Seeds were grown under chemically defined conditions; the culture medium and supernatant, free of animal components, were collected by centrifugation and subsequent filtration. Purification was achieved using protein A affinity chromatography (MabSelect SuRe, Cytiva). The pH of HCCF was adjusted to 7.5, and the column was equilibrated with binding buffer (20 mM NaH2PO4, 50 mM NaCl, 1 mM EDTA, pH 7.5). The flow rate was adjusted according to the culture titration to achieve optimal retention time on the column. Emulsion was performed in the elution buffer (100 mM trisodium citrate, pH 3.0). After neutralization of the extract and buffer exchange to storage buffer (PBS, 100 mM L-arginine), the antibody was stored at 8 to 15 mg / mL at 2 to 8°C (short-term storage) or rapidly frozen and stored at -80°C (long-term storage). The Tub-tag conjugation reaction of belentoxicin was performed using TTL.

[0301] TTL catalytic ligation was performed according to previous literature (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791, WO 2016 / 066749, WO 2017 / 186855). Briefly, 3-methoxy-L-tyrosine was ligated to belentoxigenic Tub-tag monoclonal antibody in various volumes consisting of 20 mM MOPS / KCl, 100 mM KCl, 10 mM MgCl2, 5 mM ATP, 5 mM 3-methoxy-L-tyrosine, and pH 7.0. After pH adjustment, belentoxigenic Tub-tag and TTL were added, and the reaction was carried out at 18°C ​​for 12 hours to tyrosinate the antibody. Purification of tyrosinated belentoxigenic Tub-tag

[0302] The crude tyramine acidification reaction was diluted to reduce conductivity (<10 mS / cm). The mixture was then loaded into a 5 mL HiTrap Capto Q ImpRes column (Cytiva). After washing the column with 5 CV AEX binding buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, pH 4.5) and 5 CV 10% AEX elution buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 1 M NaCl, pH 4.5), tyramine-treated belentoxigenic tub-tag was eluented with a linear gradient of more than 10 CV to 50% elution buffer. Protein concentrations containing differentiated pooled products were measured using the UV280 method (NanoPhotometer NP80, IMPLEN). Oxime ligation after TTL reaction.

[0303] Adjust the protein concentration of tyramine-modified belentoxigenic Tub-tag purified by anion exchange chromatography and add DMSO as a co-solvent (finally 2% v / v). Oxime ligation was performed at 18°C ​​using a slight excess of 2(HA-vc-PAB-MMAE) or 3(HA-(VC-PAB-MMAE)2) until the reaction was complete. The structures of effective loads 2 and 3 are described in Scheme 2. Purification of belentoxigenic Tub-tag ADC

[0304] The crude oxime-ligand was diluted with CEX binding buffer (1 to 1 v / v, 14.4 mM Na2HPO4, 5.6 mM NaH2PO4, pH 4.5) and loaded into a 5 mL HiTrap Capto S ImpAct column (Cytiva). After washing the column with 10 CV of CEX wash buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 0.1% Triton-x-114, pH 4.5) and 5 CV of CEX binding buffer, TUB-010 was extracted using a linear gradient of over 20 CV to 100% CEX extraction buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 1 M NaCl, pH 4.5). The pH of the reservoir was adjusted to 7.2. Buffer was prepared by buffer exchange via TFF (Vivaflow 50R). Synthesis of 3-methoxy-L-tyrosine: N-[(tert-butoxy)carbonyl]-L-tyrosine

[0305] To a solution of 20 g L-tyrosine (0.110 mol, 1 equivalent) in 300 ml H2O and 300 ml 1,4-dimethylamine, 24.5 ml triethylamine (0.176 mol, 1.6 equivalent) was slowly added. The reaction mixture was cooled to 0°C, and 26.4 g dibutyl dicarbonate (0.121 mol, 1.1 equivalent) was added in portions. The reaction mixture was then slowly heated to room temperature overnight. The organic solvent was removed under reduced pressure, and the solution was acidified to pH 1 with 2N HCl. The aqueous phase was extracted three times with 200 ml EtOAc, the organic components were combined, dried over MgSO4, and all volatiles were removed under reduced pressure. The desired compound was given as a white solid (30.6 g, 0.109 mmol, 99%). The analytical data are consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791). N-[(tert-butoxy)carbonyl]-L-methyltyrosine

[0306] To a suspension of 20 g N-[(tributoxy)carbonyl]-L-tyrosine (0.071 mol, 1 equivalent) in 300 ml chloroform, 2.56 ml H2O (0.142 mol, 2 equivalents) and 17.1 mg powdered sodium hydroxide (0.427 mol, 6 equivalents) were added in portions. The reaction mixture was heated under reflux overnight with vigorous stirring. The mixture was then cooled to room temperature and diluted with 300 ml H2O. The organic layer was separated and discarded. The aqueous layer was acidified to pH 1 with 2N HCl and extracted three times with 200 ml EtOAc. The organic components were combined, dried over MgSO4, and all volatiles were removed under reduced pressure. The crude product was purified by silica column chromatography (5% MeOH / CH2Cl2 + 0.5% formic acid) to give a pale yellow solid (5.26 g, 0.017 mol, 24%). The analytical data were consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791). L-methoxytyrosine TFA salt

[0307] Suspend 500 mg of N-[(tributoxy)carbonyl]-L-methoxytyrosine (1.616 mmol, 1 equivalent) in 1 ml of CH2Cl2 and cool to 0°C. Add 0.1 ml of H2O, then add 1 ml of trifluoroacetic acid dropwise. The reaction mixture was stirred at 0°C for 3 hours, and all volatiles were removed under a nitrogen stream. The crude product was purified by preparative HPLC (Gilson Inc) using a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH) with the following gradient: (A = H₂O + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 30 mL / min, 5% B 0 to 5 min, 5 to 30% B 5 to 50 min, 99% B 50 to 60 min). After lyophilization, the desired product, TFA salt, was obtained as a pale yellow powder (397 mg, 1.228 mmol, 76%). The analytical data were consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791). Hydroxylamine- Synthesis of MMAE (HA-VC-PAB-MMAE) (Fmoc-aminooxyacetic acid (FMOC-HA-COOH))

[0308] In a 50 ml round-bottom flask, add 100 mg (0.915 mmol, 1.0 equivalent) of O-(carboxymethyl)hydroxylamine hemihydrochloride (Sigma-Aldrich) dissolved in 4 ml of water in portions, and 240 mg (2.264 mmol, 2.1 equivalent) of sodium carbonate. Cool the solution to 0°C and add dropwise 260 mg (1.005 mmol, 1.1 equivalent) of methoxycarbonyl chloride (TCI-chemicals) in 2 ml of 1,4-dimethylalkane. Warm the solution to room temperature overnight. Remove the dimethylalkane under reduced pressure, add 50 ml of water, and acidify the solution with 2 mol / L HCl. Extract the aqueous phase three times with ethyl acetate, and dry the combined organic phases with MgSO4. Remove all volatiles under reduced pressure to obtain the desired compound as a white solid. The analytical data (283 mg, 99.0%) were consistent with previously reported results (Clavé et al. (2008), Organic & Biomolecular Chemistry, 6:3065-3078). Fmoc-VC-PAB-PNP

[0309] 200 mg Fmoc-VC-PAB (0.333 mmol, 1.0 equivalent), 202 mg bis(4-nitrophenyl) carbonate (0.333 mmol, 1.0 equivalent), and 1 ml DMF were added to a 10 ml screw-cap vial. 232 μl DIPEA (1.332 mmol, 4.0 equivalent) was added, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was poured into ice-cold diethyl ether, and the filtrate was collected by centrifugation. The filtrate was redissolved in 1 ml DMF, and precipitated a second time with ice-cold diethyl ether. The precipitate was collected by centrifugation, dried under vacuum, and the desired product was obtained as a pale yellow powder (247 mg, 97.0%). The analytical data were consistent with previously reported results (WO2004010957). H2N-VC-PAB-MMAE

[0310] Add 60 µl of a 1 mol / L MMAE TFA salt solution in DMSO (50 mg, 0.06 mmol, 1.0 equivalent), 180 µl of a 0.4 mol / L Fmoc-VC-PAB-PNP solution in DMSO (0.072 mmol, 1.2 equivalent), and 60 µl of a 1 mol / L hydroxybenzotriazole hydrate solution in DMSO (0.06 mmol, 1.0 equivalent) to a 10 mL screw-cap vial. Add 105 µl of DIPEA (0.6 mmol, 10.0 equivalent) and stir the yellow solution at room temperature for 3 hours. After the MMAE feedstock has been completely consumed as monitored by UPLC / MS, add 120 µl of a 50% (w / w) diethanolamine solution in DMSO and stir the yellow solution for another 1 hour. Add 1.5 ml acetonitrile and 3 ml water, and purify the solution by preparative HPLC (Gilson Inc) using a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH) with the following gradient: (A = H2O + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 30 ml / min, 30% B 0 to 5 min, 30 to 60% B 5 to 25 min, 99% B 25 to 35 min). After lyophilization, the desired product, TFA salt, was obtained as a white powder (29.5 mg, 39.7%). The analytical data were consistent with previously reported results (Tang et al. (2016), Organic & Biomolecular Chemistry, 14(40): 9501-9518). HA-VC-PAB-MMAE

[0311] Add 23.3 mg H2N-VC-PAB-MMAE (0.0188 mmol, 1.0 equivalent), 38 µl of 0.6 mol / L Fmoc-HA-COOH solution in DMSO (0.0226 mmol, 1.2 equivalent), and 38 µl of 0.6 mol / L Pybop solution in DMSO (0.0226 mmol, 1.2 equivalent) to a 10 mL screw-cap vial. Add 33 µl DIPEA (0.1880 mmol, 10.0 equivalent) and stir the solution at room temperature for 2 hours. Add 150 µl of 50% (w / w) diethanolamine solution in DMSO and stir the solution further for 1 hour. Add 1.5 ml of acetonitrile and 3 ml of water, and purify the solution using a preparative HPLC system (Gilson Inc) with a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH). The gradient is as follows: (A = H2O + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 30 ml / min, 30% B 0 to 5 min, 30 to 99% B 5 to 60 min, 99% B 60 to 70 min). After freeze-drying, the desired product, TFA salt, is obtained as a white powder (20.1 mg, 81.6%). HR-MS determination of C60H98N11O14+[M+H]+: calculated value: 1196.7290, measured value: 1196.7278. The HPLC chromatogram of the purified substance is shown in Figure 15. The synthesis of branched-chain MMAE hydroxylamine HA-(VC-PAB-MMAE)2: BOC-Glu(OSu)2 was synthesized by adding 1.00 g Boc-Glu-OH (4.04 mmol, 1.00 equivalent), 0.93 g N-hydroxybutyric acid diimide (8.08 mmol, 2.00 equivalent), and 25 mg DMAP (0.20 mmol, 0.05 equivalent) dissolved in 20 mL anhydrous THF to a 50 mL round-bottom flask. The suspension was cooled to 0 °C, and 1.84 g dicyclohexanediimide (8.88 mmol, 2.2 equivalent) dissolved in 10 mL anhydrous THF was added dropwise. The white suspension was warmed to room temperature overnight. The mixture was filtered, the filtrate was evaporated, and the residue was purified by silica gel rapid column chromatography. The desired product was obtained as a colorless oil (460 mg, 1.04 mmol, 25.8%). The analytical data are consistent with previously reported results (Koshi et al., J. Am. Chem. Soc. 2008, 130, 1, 245–251). Boc-Glu-(VC-PAB-MMAE)2

[0312] Add 15.83 mg H2N-VC-PAB-MMAE TFA salt (0.012 mmol, 2.00 equivalent), 2.84 mg Boc-Glu(OSu)2 (0.06 mmol, 1.00 equivalent), and 150 µL DMF to a 10 ml screw-cap vial. Add 4.5 µL DIPEA (0.0256 mmol, 4.0 equivalent) and stir the solution at room temperature for 2 hours. Add 1.5 ml of acetonitrile and 3 ml of water, and purify the solution using a preparative HPLC system (Gilson Inc, WI, Middleton, USA) with a VP 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) at a Gilson PLC 2020 system (Gilson Inc, WI, Middleton, USA). The gradient is as follows: (A = H2O + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 10 ml / min, 30% B 0 to 5 min, 30 to 99% B 5 to 60 min, 99% B 60 to 70 min). After lyophilization, the desired product is obtained as a white powder. (8.84 mg, 0.0036 mmol, 55.8 mg / mL) %). HR-MS determination of C126H2O3N21O282+[M+2H]2+: Calculated value: 1229,7565, Measured value: 1229,7567. The HPLC chromatogram of the purified substance is shown in Figure 16. Boc-HA-Glu-(VC-PAB-MMAE)2

[0313] Add 3.99 mg Boc-Glu-(VC-PAB-MMAE)2 (1.63 µmol, 1.00 equivalent) to a 10 ml screw-cap vial, dissolve in 100 µL CH2Cl2, and cool to -20°C. Add 100 µL trifluoroacetic acid dropwise. After 3 hours at -20°C, raise the temperature to 0°C and remove all volatiles in a stream of N2. Add 0.94 mg Boc-HA-OSu (3.26 µmol, 2.00 equivalent) dissolved in 100 µL DMF, then add 2.8 µl DIPEA (0.163 µmol, 10.0 equivalent), and stir the solution at room temperature for 2 hours. Add 1.5 ml of acetonitrile and 3 ml of water, and purify the solution using a preparative HPLC system (Gilson Inc, WI, Middleton, USA) with a VP 250 / 10 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) at a gradient of: (A = H2O + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 5 ml / min, 30% B 0 to 5 min, 30 to 99% B 5 to 60 min, 99% B 60 to 70 min). After lyophilization, the desired product was obtained as a white powder. (2.11 mg, 0.748) µmol (46%). HR-MS determination of C128H2O6N22O3O2+[M+2H]2+: Calculated value: 1266, 2647; Measured value: 1266, 2644. The HPLC chromatogram of the purified substance is shown in Figure 17. HA-(VC-PAB-MMAE)2

[0314] Add 2.11 mg of Boc-Glu-(VC-PAB-MMAE)2 (0.75 µmol) to a 10 ml screw-cap vial, dissolve in 100 µL of CH2Cl2, and cool to -20°C. Add 100 µL of trifluoroacetic acid dropwise. After 3 hours at -20°C, raise the solution to 0°C and remove all volatiles in a stream of N2. The desired product was obtained as a white powder after freeze-drying in 50% MeCN / H2O (1.90 mg, 0.748 µmol, quantitative). HR-MS determination of C123H198N22O282+[M+2H]2+: Calculated value: 1216, 2384; Measured value: 1216, 2326. The HPLC chromatogram of the purified substance is shown in Figure 18. Rapid and thin-layer chromatography methods.

[0315] Rapid column chromatography was performed using NORMASIL 60® silicone 40 to 63 µm (VWR International, USA). Glass TLC plates coated with fluorescence indicator F254s were used. Silicone 60 W was purchased from Merck (Merck Group, Germany). Spots were observed by fluorescence depletion under a 254 nm lamp or staining with manganese (10 g K2CO3, 1.5 g KMnO4, 0.1 g NaOH in 200 ml H2O), followed by heating. Preparative HPLC

[0316] Preparative HPLC was performed on a Gilson PLC 2020 system (Gilson Inc, WI, Middleton, USA) using a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany). Use the following gradients: Method C: (A = H₂O + 0.1% TFA (trifluoroacetic acid), B = MeCN (acetonitrile) + 0.1% TFA, flow rate 30 ml / min, 5% B 0 to 5 min, 5 to 90% B 5 to 60 min, 90% B 60 to 65 min. Method D: (A = H₂O + 0.1% TFA, B = MeCN + 0.1% TFA), flow rate 30 ml / min, 5% B 0 to 5 min, 5 to 25% B 5 to 10 min, 25% to 45% B 10 to 50 min, 45 to 90% B 50 to 60 min, 90% B 60 to 65 min. Method E: 0.1% TFA, flow rate 18 ml / min, 5% B 0 to 5 min, 5 to 90% B 5 to 60 min, 90% B 60 to 65 min, using VP. 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum tubing (Macherey-Nagel GmbH & Co. Kg, Germany). HR-MS

[0317] High-resolution ESI-MS spectra were recorded on a Waters Class H instrument equipped with a quaternary solvent manager, Waters sample manager-FTN, Waters PDA detector, and Waters column manager, featuring an Acquity UPLC protein BEH C18 column (1.7 µm, 2.1 mm x 50 mm). Samples were extracted at a flow rate of 0.3 mL / min. The following gradients were used: A: 0.01% FA in H2O solution; B: 0.01% FA in MeCN solution. 5% B: 0 to 1 min; 5 to 95% B: 1 to 7 min; 95% B: 7 to 8.5 min. Quality analysis was performed using a Waters XEVO G2-XS QTof analyzer. UPLC-UV / MS

[0318] UPLC-UV / MS traces were recorded on a Waters Class H instrument equipped with a quaternary solvent manager, a Waters autosampler, a Waters TUV detector, and a Waters Acquity QDa detector. The instrument featured an Acquity UPLC BEH C18 1.7 μm, 2.1 x 50 mm RP column at a flow rate of 0.6 mL / min (Waters Corp., USA). Purity analysis was performed using the following gradients: A: 0.1% TFA in H2O solution; B: 0.1% TFA in MeCN solution. 5% B 0 to 1.5 min, 5% to 95% B 1.5 to 11 min, 95% B 11 to 13 min, 5% B 13 to 15 min. Intact protein MS

[0319] Intact proteins were analyzed using a Waters Class H instrument equipped with a quaternary solvent manager, Waters Sample Manager-FTN, Waters PDA detector, and Waters column manager, featuring an Acquity UPLC protein BEH C4 column (300 Å, 1.7 µm, 2.1 mm x 50 mm). Protein was extracted at a flow rate of 0.3 mL / min using the following gradients: A: 0.01% FA in H2O solution; B: 0.01% FA in MeCN solution. 5 to 95% B 0 to 6 min. Mass analysis was performed using a Waters XEVO G2-XS QTof analyzer. Protein was ionized in positive ion mode with a cone voltage of 40 kV. Raw data were analyzed using MaxEnt 1. Resazurin assay.

[0320] HL60 and Karpas cell lines were cultured in RPMI-1640 medium supplemented with 10% FCS and 0.5% penicillin-streptomycin. Cells were seeded at a density of 5*10³ cells / well in 96-well cell culture microplates. ADC or antibody was serially diluted 1:4 in cell culture medium to a final concentration of 3 µg / ml and transferred in duplicate to each well of the microplate. The plates were incubated at 37°C and 5% CO₂ for 96 hours. Subsequently, resorufin was added to a final concentration of 50 µM, followed by incubation at 37°C and 5% CO₂ for 3 to 4 hours. The metabolic conversion of resorufin to resorufin was quantified using the fluorescence signal (λEX = 560 nm, λEM = 590 nm) of resorufin on a Tecan Infinite M1000 microplate reader. The mean and standard deviation were calculated from two copies, normalized to the untreated control group, and antibody concentration was plotted. Data analysis was performed using MATLAB R2016. ADC characterization and stability were assessed using A-SEC.

[0321] Adjust the ADC to a protein concentration of 1 mg / mL in PBS (Dulbeccos phosphate-buffered saline, Sigma-Aldrich Merck KGaA) and filter sterilize (Ultrafree-MC centrifuge filter units, Merck Millipore). Store samples at 4–8°C, 37°C, and 40°C for up to 14 days. For samples stored at high temperatures, ensure no condensation forms. Centrifuge samples at 4°C, 4000 xg for 4 minutes prior to A-SEC analysis. In vivo xenograft model

[0322] In vivo efficacy studies were conducted in accordance with animal welfare laws and approved by local authorities. In short, Karpas 299 cells were subcutaneously injected into CB17-Scid mice on day 0. Treatment began when the tumor reached a certain average tumor volume. After randomization to treatment and control groups, the ADC was administered intravenously. Tumor volume, body weight, and general health status were recorded throughout the study. Pharmacokinetic analysis was performed.

[0323] Experiments were conducted in accordance with animal welfare laws and with approval from local authorities. On day 1, the test substance was administered to Sprague Dawley rats via a single intravenous (bolus) injection. For bioanalysis, blood was collected at various time points via the jugular vein in a target volume of 1 mL over a period of up to 504 hours. Samples were allowed to coagulate at room temperature for at least 1 hour. Samples were then centrifuged at 1500 g and 4°C for 10 minutes. The resulting serum was stored at -20°C. PK parameters were calculated using Phoenix (WinNonlin) pharmacokinetic software 1.4 (Certara, 6.4), employing a non-compartmental method consistent with the intravenous (bolus) administration route, and an in vivo curve-forming method (extravascular) was used for the free payload. Repeated-dose toxicity studies were conducted.

[0324] Experiments were conducted in accordance with animal welfare laws and with approval from local authorities. Male and female Stowe rats (10 rats / sex / group) were administered weekly (days 1, 8, 15, and 22) with a control group (mediator), belentuximab Tub-tag ADC (10 mg / kg), or belentuximab vedotin (10 mg / kg). Autopsy was performed on day 26 during the main dosing period (5 rats / sex / group) and on day 51 during the recovery period (5 rats / sex / group). Clinical observations, body weight, and food consumption were measured daily throughout the study. Pharmacokinetic samples were obtained from each animal before each dosing, 15 minutes after each dosing, and at autopsy. All animals were autopsied, organs were weighed, and preserved in fixatives. Clinicopathological (hematological, clinical chemistry, and coagulation endpoint) samples were also collected at autopsy. Example 1: Belentuximab Tub-tag ADC for the treatment of CD30+ indications

[0325] Tub-tag technology utilizes a natural α-tubulin-derived peptide, which is highly polar and serves as the recognition sequence for the enzyme tubulin tyrosine ligase (TTL), which is the recognition sequence of tubulin tyrosine ligase as disclosed in this paper.

[0326] TTLs recognize short, hydrophilic peptide tags (Tub-tags) and catalyze the formation of peptide bonds with tyrosine derivatives and amino acid-like structural units. Here, the inventors demonstrate that Tub-tag technology can be used to conjugate the payload with the antibody belentoxicin (cAC10), promoting CD30 binding to the ADC, and remarkably outperforming the approved belentoxicin-based ADC belentoxicin vedocinine in terms of stability, efficacy, and toxicology. Method 1: Using Tubulis' proprietary Tub-tag® ("TTL recognition sequence") technology, the drug moiety is site-specifically fused to a monoclonal antibody (such as belentoxicin) via a linker (payload). TTL: Tubulin tyrosine ligase.

[0327] The inventors initiated their research by fusing the recognition sequence of a tubulin tyrosine ligase with the light chain (light chain SEQ ID NO: 11, heavy chain: SEQ ID NO: 1), heavy chain (light chain SEQ ID NO: 12), or light chain and heavy chain (light chain SEQ ID NO: 11, heavy chain: SEQ ID NO: 12), or light chain and heavy chain (light chain SEQ ID NO: 11, heavy chain: SEQ ID NO: 12) of the monoclonal antibody cAC10 (belentoxigenic, exemplarily shown in Scheme 2 as a Tub-tag fused with the light chain) (at the nucleic acid level), optionally with a sequence of a Gly4Ser amino acid linker arranged between the recognition sequence of the tubulin tyrosine ligase and the corresponding antibody chain. Scheme 2: Chemical enzyme conjugation for ADC manufacturing. (Blue) Tub-tag recognition sequence, (black) tyrosine derivative, (yellow) payload with a bio-orthogonal stalk. Tub-tags can be placed on the light and / or heavy chains to obtain homogeneous ADCs with drug-to-antibody ratios (DARs) ranging from 2 to 8, depending on the number of Tub-tags used. Loads 2 and 3 are also described. These belentoxigenic variants were recombinantly expressed using established cell culture methods, followed by successful purification by protein A chromatography. Particle size sieving (SEC) analysis showed that the Tub-tag antibodies were of very high purity, with only very small amounts of high molecular weight substances (HMWS) observed (HMWS = 1.5% to 7%). The results are shown in Figure 1. These results indicate that the antibodies are highly monomeric after expression and purification. As shown in Figure 1B, the attachment of the Tub-tag recognition sequence to the belentoxigenic heavy chain resulted in a 10-fold reduction in HMWS from 11% to only 1.5%. Figure 1A shows the particle size distribution of unmodified belenutoxine after protein A chromatography (PAC), which is referred to herein simply as "Bren. HC-Tub". "HMWC" stands for "High Molecular Weight Component". Figure 1B shows the particle size distribution of belenutoxine after protein A chromatography (PAC) containing a TTL recognition sequence (Tub-tag) fused to the heavy chain (light chain: SEQ ID NO: 2, heavy chain: SEQ ID NO: 12), which is also referred to herein as "Bren. HC-Tub". Figure 1C shows the particle size distribution of belenutoxine after protein A chromatography (PAC) containing a TTL recognition sequence (Tub-tag) fused to the light chain (light chain: SEQ ID NO: 11, heavy chain: SEQ ID NO: 1), which is also referred to herein as "Bren. LC-Tub".Figure 1D shows the particle size sieving chromatography of berentuximab after protein A chromatography (PAC). This berentuximab contains a TTL recognition sequence (Tub-tag) fused with the light chain and the heavy chain (light chain: SEQ ID NO: 11, heavy chain: SEQ ID NO: 12), which is also referred to herein as "Bren. LCHC-Tub".

[0328] After demonstrating the manufacturability of belentoxigenin containing a microtubule-tyrosine ligase recognition sequence (“Tub-tag variant” or “belentoxi Tub-tag”) at the C-terminus of the light chain, heavy chain, or all heavy and light chains of belentoxigenin, the inventors determined the retention time using hydrophilic interaction chromatography (HIC), a commonly used measure of hydrophobicity. This study revealed that all Tub-tag variants exhibited higher hydrophilicity and surface polarity compared to unmodified cAC10 (belentoxigenin) (Figure 2A). Figure 2A shows analytical hydrophobic interaction chromatography for belentoxigenin (black), Bren. LC-Tub (grey, dashed line), Bren. HC-Tub (grey), and Bren. LCHC-Tub (black, dashed line). The chromatography shows the normalized absorption spectra recorded at 220 nm. Retention time is a measure of hydrophobicity. Longer retention times in hydrophobic interaction chromatography indicate greater hydrophobicity, while shorter retention times indicate greater hydrophilicity. Since alterations to the antibody backbone can perturb structure and stability, differential scanning fluorometry (DSF) was used to measure the melting points (Tm) of belentoxicin and the Tub-tag variants. These experiments confirmed that the Tub-tag variants have the same melting point (Tm = 70.9–71.8 °C) as the unmodified monoclonal antibody, and therefore exhibit the same thermal stress stability (Tm = 71.0 °C) (Figure 2B). Figure 2B shows the differential scanning fluorometry curves used to determine the melting points (Tm) of belentoxicin, Bren. LC-Tub, Bren. HC-Tub, and Bren. LCHC-Tub.

[0329] The inventors tested the enzymatic modification of Tub-tag antibodies with different tyrosine derivatives, and 3-methoxy-L-tyrosine was proven to be the optimal choice for ADC fabrication. In short, the inventors co-cultured different belentoxigenic Tub-tag variants with TTL to covalently link 3-methoxy-L-tyrosine to the terminal glutamic acid residues of the Tub-tag. Specifically, the amino group of 3-methoxy-L-tyrosine was linked to the C-terminus of the Tub-tag. Excess 3-L-methoxy-tyrosine and residual TTL were then removed by protein A chromatography or anion exchange chromatography (AEX). Subsequently, the linker-particle load ("linker-drug moiety") was covalently linked by a bioorthogonal reaction. The resulting ADC was purified using cation exchange chromatography (CEX) to remove excess linker particle load and improve ADC purity. The final step of this method involves formulation via tangential flow filtration (TFF) and aseptic filtration. To produce an ADC with a drug-to-antibody ratio (DAR) of 2, we use linear payload 2 (as shown in Scheme 2, "structure 4" conjugated with MMAE as defined in the specification and claims). To produce an ADC with a DAR of 4, we use branched payload 3 (as shown in Scheme 2, "structure 5" conjugated with MMAE as defined in the specification and claims). Specifically, the H2N-O- portions of payloads 2 and 3 react with the formyl group of 3-cresyl-L-tyrosine bound to the TTL recognition sequence to obtain an oxime having a -C=NO- structure. This procedure yields pure, homogeneous ADCs, with the quality controlled, for example, by SEC, HIC (Figure 3), and intermediate MS (Figure 4), demonstrating excellent purity and homogeneity. The following ADCs were prepared and further investigated in examples (payloads 2 and 3 are shown in Scheme 2): ADC Berentoxi light chain Berentoxice Heavy Chain Non-natural amino acids Payload Drug-to-antibody ratio (DAR) Bren. HC-2 SEQ ID NO: 2 SEQ ID NO: 12 (Includes TTL identification sequence) 3-Methyl-L-tyrosine Payload 2 2 Bren. LC-2 SEQ ID NO: 11 (Includes TTL identification sequence) SEQ ID NO: 1 3-Methyl-L-tyrosine Payload 2 2 Bren. LCHC-2 SEQ ID NO: 11 SEQ ID NO:12 3-Methyl-L-tyrosine Payload 2 4 Bren. LC-3 SEQ ID NO: 11 SEQ ID NO: 1 3-Methyl-L-tyrosine Payload 3 4 Figure 3 illustrates the analysis of belentoxigenic Tub-tag ADCs via SEC (Figures 3A to 3C) and HIC (Figures 3D to 3F). Figures 3A and 3D show the results for Bren. HC-vc-PAB-MMAE DAR2 (also referred to herein as Bren. HC-2), Figures 3B and 3E show the results for Bren. LC-vc-PAB-MMAE DAR2 (also referred to herein as Bren. LC-2), and Figures 3C and 3F show the results for Bren. LCHC-vc-PAB-MMAE DAR4 (also referred to herein as Bren. LCHC-2). Each belentoxigenic variant was expressed, purified with PAC, and conjugated with payload 2 to produce DAR2, and conjugated with either payload 2 or payload 3 to produce DAR4 ADC. After final polishing with HIC and buffer exchange, the Tub-tag ADCs were analyzed based on aggregate content and DAR homogeneity. All Tub-tag ADCs contained very low HMWS (<1%) and exhibited excellent DAR homogeneity. Figure 4 shows the analysis of berentoxigenic Tub-tag ADCs by intermediate protein MS after deglycosylation. The deconvolution spectra are shown. Figure 4A shows the results for Bren. HC-Tub; Figure 4B shows the results for Bren. LCHC-Tub; Figure 4C shows the results for Bren-LC-Tub; Figure 4D shows the results for Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"); Figure 4E shows the results for Bren-LCHC-vc-PAB-MMAE (also referred to herein as "Bren. LCHC-2"); and Figure 4F shows the results for Bren. LC-2(vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"). The mass shift of 1369 m / z is due to the incorporation of 3-methoxy-L-tyrosine and the linkage of the oxime to payload 2. A mass difference of 2604 m / z was observed after the oxime was linked to payload 3. These results are consistent with the calculated values. The sensitivity of payloads 2 and 3 to cathepsin B cleavage was analyzed before conjugation with the antibody (Figure 5). The results show that payloads 2 and 3 were cleaved by cathepsin B, releasing free monomethylarrestatin E (MMAE). Figure 5 shows the RP-HPLC analysis of payload 2 containing valine-citrulline (VC) during the digestion reaction with cathepsin B (1:1000 for each VC fraction) shown in Figure 5A. The reaction was terminated with E-64 before analysis. Chromatography was recorded at 220 nm after a reaction time of 150 min. Figure 5B shows the increase (black) of free MMAE and the decrease (gray, circles) of payload 2 over time during the reaction with cathepsin B. Figure 5C shows the pyrolysis of payload 3.During the reaction of payload 3 with cathepsin B, an intermediate (gray, triangular) containing an MMAE moiety is formed. Example 2: In vitro stability of the ADC.

[0330] To clarify the stability of the belentoxicin ADC described in Example 1, the inventors performed a thermal stress test based on the fluorescent dye SYPRO Orange. The DSF experiment demonstrated that the belentoxicin Tub-tag ADC has a melting point similar to the corresponding monoclonal antibody (Blentoxicin Tm = 71.0 °C, Belentoxicin Tub-tag ADC Tm = 70.9–72.9 °C). This demonstrates the antibody-like stability of the belentoxicin Tub-tag ADC. The belentoxicin vedotin used for comparison showed a lower melting point of 68.5 °C, and the melting curve covered a higher temperature range. Furthermore, the stability of the ADC was studied in buffer and mouse plasma. Samples stored in buffer were separated by SEC to analyze the formation of HMWS (high molecular weight substances). After two weeks of storage at 4 °C, no significant changes were observed in the chromatogram. However, the differences became apparent during high-temperature storage. After dissolving belentoxicin vedotin according to the manufacturer's instructions, the HMWS was measured to be 0.74%. During 14 days of storage at 40°C, the value increased to 11.44%, representing a 15.5-fold increase in HMWS. In comparison, belenutoxicillin Tub-tag ADC was very stable, with a low-fold increase in HMWS of 1.0–5.0-fold (Figure 6A). Although belenutoxicillin LC-3DAR 4 contains the same drug load as belenutoxicillin (DARav4) and therefore exhibits the high hydrophobicity derived from the effective load, a low-fold increase in HMWS was observed. After manufacturing Tub-tag ADC Bren. LC-3 with DAR 4, the measured HMWS was 0.47%. During storage at 40°C, only a slight increase in HMWS was observed to 2.34% (Figure 6C; results for belenutoxicillin are shown in Figure 6B for comparison). Figure 6 shows the storage results of belenutoxicillin and belenutoxicillin Tub-tag ADC at high temperatures. Figure 6A shows the increase in HMWS during the study (Bren. LC-2(vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"); Bren. LC-vc-PAB-MMAE (also referred to herein as "Bren. LC-2"); Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"); and belentuxevidoline. Belentuxevidoline showed the greatest increase in aggregate content. In contrast, the HMWS content of the Tub-tag ADC remained almost unchanged. Figures 6B and 6C show SEC chromatography, which show the normalized absorption spectra of belentuxevidoline (mean drug-to-antibody ratio (DARav) 4) and Bren. LC (Bren. LC-3, DAR 4) conjugated with payload 3 at 220 nm.

[0331] Furthermore, the stability of belentuximab Tub-tag ADCs in mouse plasma has been evaluated, showing excellent stability. In contrast, belentuximab vedotin was rapidly degraded (Figure 7, Table 1). The rapid degradation of maleic anhydride-linked ADCs (such as belentuximab vedotin) has recently been shown, and reverse mycoaddition may be an explanation for our observation. Table 1: Storage of belentuximab vedotin and belentuximab Tub-tag ADCs in mouse plasma for 7 days at 37°C. ADC DAR before plasma culture DAR after plasma culture av n=2 Payload loss [%] Bren. HC-2 2 1.8 10 Bren. LC-2 2 1.8 10 Bren. LCHC-2 3.8 3.63 5 Bren. LC-3 4 3.59 10 Berentoxividotin 4.02 1.53 62 Figure 7 illustrates an exemplary description of the stability of belentuximab-Tub-tag ADC in mouse plasma compared to belentuximab-Vidodine. Figures 7A and 7B show the results of storing belentuximab-Vidodine in mouse plasma at 37°C. Figures 7C and 7D show the results of storing Bren. LC-3DAR 4 in mouse plasma at 37°C. Example 3: In Vitro Efficacy of ADC

[0332] Next, the inventors evaluated the in vitro efficacy of belentuximab Tub-tag ADC in a cell viability assay and compared the results with those of belentuximab vedotin. The CD30-overexpressing cell line Karpas299 and the CD30-negative cell line HL60 were used in this resplenoid assay (Figure 8). The IC50 value of belentuximab vedotin (IC50 = 2.08) measured in the antigen-positive cell line Karpas299 was consistent with literature data. Overall, belentuximab Tub-tag ADC showed a low IC50 value and good in vitro efficacy (Table 2). Table 2: IC50 values ​​of belentuximab vedotin and belentuximab Tub-tag ADC determined by resplenoid assay. ADC IC 50 Karpas299 cells Bren. HC-2 5.24 n=1*2 Bren. LC-2 5.63 n=4*2 Bren. LCHC-2 1.70 n=2*2 Bren. LC-3 1.06 n=5*2 Berentoxividotin 2.08 n=5*2 Figure 8 shows the in vitro efficacy of belentoxigenic Tub-tag ADC in the CD30-overexpressing cell line Karpas299 and the CD30-negative cell line HL60. Example 4: In vivo efficacy of belentoxigenic Tub-tag ADC

[0333] In vivo efficacy of belentuximab Tub-tag ADCs was evaluated in immunodeficient CB17-SCID mice using a Karpas299-derived tumor model. As shown in Figures 9 and 10, Bren. LC-2 exhibited unexpectedly high in vivo efficacy similar to belentuximab vedocinine. This observation was made despite belentuximab LC-2 having half the drug load, as it is a DAR 2 MMAE ADC compared to belentuximab vedocinine which is conjugated to 4 MMAE molecules. Additionally, unexpectedly, Bren. LC-3 significantly outperformed belentuximab vedocinine in vivo efficacy, with a median survival increase of 2.5-fold (Figure 11). Figure 9 shows the in vivo efficacy of Bren. LC-2 expressed as tumor volume (cm3). To evaluate the in vivo efficacy of Bren. LC-2, mice with tumor volumes between 100 and 150 mm3 were randomly assigned and treated with a single injection of 1.5 mg / kg on days 7 and 10 post-tumor transplantation. Figure 10 shows the in vivo efficacy of Bren. LC-2, expressed as tumor volume (cm3) and survival percentage of treated animals (Kaplan-Mail plot). To evaluate the in vivo efficacy of Bren. LC-2, mice with tumor volumes between 100 and 150 mm3 were randomly assigned and treated with a single injection of 1.0 mg / kg. Figure 11 shows the in vivo efficacy of Bren. LC-3, expressed as tumor volume (cm3) and survival percentage of treated animals. To evaluate the in vivo efficacy of Bren. LC-3, mice with tumor volumes between 100 and 150 mm3 were randomly assigned and treated with a single injection of 0.5 mg / kg on days 8 and 11 post-tumor transplantation. Example 5: Pharmacokinetics of Berentoxicin Tub-tag ADC

[0334] Inspired by the remarkable discovery that belenutoxicillin Tub-tag ADC exhibits high in vitro stability and excellent in vivo efficacy, the inventors sought to elucidate whether this in vitro stability translates into living organisms and conducted pharmacokinetic analysis in Stuart's rats. On day 1, the test substance was administered to the animals via a single intravenous (bolus) injection. To analyze ADC levels over time, an ELISA-based assay was developed to detect antibodies and intact ADC (by measuring the effective load bound to the antibody). The total antibody assay provides useful information about antibody clearance, and the difference in detection compared to the intact ADC assay explains the loss of antibody effective load. Additionally, the inventors developed an MS-based assay, which facilitates the measurement of effective load loss and transfer of effective load to blood proteins. Studies have shown that belenutoxicillin Tub-tag ADC exhibits excellent in vivo stability (Figure 12A). Furthermore, compared to belenutoxicillin vedotin, belenutoxicillin Tub-tag ADC does not possess the ability to transfer effective load and covalently bind to blood proteins (Figure 12B). Figure 12 shows the pharmacokinetic (PK) analysis of belentoxigenic Tub-tag MMAE (Bren LC-2). Figure 12A shows the amount of intact ADC compared to belentoxigenic vitoline. Figure 12B shows the amount of MMAE transferred to blood proteins as analyzed by MS-analysis. Example 6: Repeated-dose toxicity study of TUB-010

[0335] To date, belenutoxicillin Tub-tag ADC has been shown to have increased in vivo efficacy and stability. Therefore, the inventors wanted to know whether increased in vivo stability resulted in beneficial toxicological properties of belenutoxicillin Tub-tag ADC. This is advantageous because the combination of enhanced toxicological properties and enhanced efficacy can significantly broaden the therapeutic range and thus offer great benefits to patients. Therefore, the inventors evaluated the toxicity and toxicokinetics of belenutoxicillin Tub-tag ADC in male and female Stuart's rats and compared these properties with those of belenutoxicillin vedocin. Male and female Stuart's rats were administered weekly (on days 1, 8, 15, and 22) as a control (mediator), belenutoxicillin Tub-tag ADC (Bren LC-2), or belenutoxicillin vedocin (10 mg / kg). Autopsy was performed on day 26 during the main dosing period (5 rats / sex / group) and on day 51 during the recovery period (5 rats / sex / group). Throughout the study, clinical observations, body weight, and food consumption were measured daily. Pharmacokinetic samples were obtained from each animal before each administration, 15 minutes after each administration, and at necropsy. All animals were necropsed, organs were weighed, and preserved in fixatives. Clinicopathological (hematological, clinical chemistry, and coagulation endpoint) samples were also collected at necropsy. The mean serum concentrations of intact ADC and total antibodies are shown in Figure 13. This figure clearly demonstrates that administration of Bren LC-2, even with repeated dosing, results in reasonable serum concentrations in vivo. The high overlap between intact ADC and total antibodies from Bren LC-2 further highlights the excellent in vivo stability of the construct and distinguishes it from the belentuximab Tub-tag group. Figure 13 shows the mean serum concentrations of intact ADC and total antibodies in male and female rats after intravenous (bolus) administration of belentuximab Tub-tag ADC at days 1, 8, 15, and 22, compared to belentuximab Tub-tag.

[0336] Unlike belentuximab vedotin, no effect on erythrocyte populations was observed with belentuximab Tub-tag ADC (Bren LC-2). This offsets the anemia (and potential thrombocytopenia) observed with belentuximab vedotin in clinical practice. Both Bren LC-2 and belentuximab vedotin were observed to induce expected MMAE-driven testicular changes, but these were delayed in Bren LC-2, where the stability of belentuximab Tub-tag ADC may play a role in the delayed onset of testicular toxicity. Exemplary toxicity characteristics of Bren LC-2 are shown in Figure 14. Figure 14 shows exemplary toxicity characteristics of belentuximab Tub-tag ADC (Bren LC-2) (right bar) in male and female rats compared to belentuximab vedotin (left bar). The parameters for the belenutoxicillin Tub-tag ADC Bren LC-2 described in Figure 14 are reticulocyte count / µL (RET, in K / µL), red blood cell count / µL (RBC, in M / µL), heme count / µL (Hb, in M / µL), hematocrit percentage (HTC, in %), eosinophil count / µL (EOS, in K / µL), activated partial clotting time (APPT) (in seconds), glucose concentration / L (in mmol / L), thymus weight (in g), and testicular weight (in g). For reference, rats were treated as a control group (mediator) (data not shown). Specifically, Figure 14 shows: Reticulocytes: Treatment with belenutoxicillin had a significant effect on reticulocyte count, while no effect was observed with treatment with belenutoxicillin Tub-tag ADC. Therefore, compared to treatment with belentuxivitoline (consistent with data on red blood cells, hemoglobin, and hematocrit), treatment with belentuxivitoline Tub-tag ADCs is expected to result in less anemia clinically. Red blood cells: Treatment with belentuxivitoline has a significant effect on red blood cell count, while no significant effect was observed with belentuxivitoline Tub-tag ADCs. Therefore, compared to treatment with belentuxivitoline (consistent with data on reticulocyte count, hemoglobin, and hematocrit), treatment with belentuxivitoline Tub-tag ADCs is expected to result in less anemia clinically. Hemoglobin: Treatment with belentuxivitoline reduces hemoglobin concentration (primarily in men), while no significant effect was observed with belentuxivitoline Tub-tag ADCs. Therefore, compared with treatment with belentoxivirdolin (consistent with data on red blood cells, reticulocytes, and hematocrit), treatment with belentoxivir Tub-tag ADC is expected to result in less anemia in clinical practice.Hematocrit: Treatment with belentuximab vedocinine reduces hematocrit (primarily in men), while no significant effect was observed with belentuximab tub-tag ADCs. Therefore, belentuximab tub-tag ADCs are expected to result in less clinically significant anemia compared to belentuximab vedocinine treatment (consistent with erythrocyte, hemoglobin, and reticulocyte data). Eosinophils: Treatment with belentuximab vedocinine results in a lower eosinophil count compared to belentuximab tub-tag ADCs. Assuming this is a general effect on white blood cells, belentuximab tub-tag ADCs are expected to result in less clinically significant neutropenia compared to belentuximab vedocinine treatment. APTT: Treatment with belentuximab vedocinine results in a lower APTT value compared to belentuximab tub-tag ADCs. Since belenutuxevidoline has no clinical effect on clotting time / APTT, given the data provided, it is also expected that belenutuxevidol ADC will not cause any concerns regarding clotting time. Glucose: Treatment with belenutuxevidoline results in higher glucose concentrations compared to treatment with belenutuxevidoline ADC. Therefore, treatment with belenutuxevidoline ADC results in lower glucose concentrations compared to treatment with belenutuxevidoline. Therefore, treatment with belenutuxevidoline ADC is unlikely to cause hyperglycemia, as has been observed clinically with belenutuxevidoline. Thymus: Treatment with belenutuxevidoline results in lower thymus weight compared to treatment with belenutuxevidoline ADC. Changes in thymus weight observed with ADCs containing MMAEs are typical of target-independent / free toxin-related toxicities caused by MMAE-induced cellular depletion. Therefore, compared to belentuximab vedoltin, belentuximab tub-tag ADCs are expected to have a more favorable clinical safety profile with fewer target-independent / free MMA-related toxicities. Testes: Treatment with belentuximab vedoltin resulted in lower testicular weight compared to treatment with belentuximab tub-tag ADCs. Changes in testicular weight observed with ADCs containing MMAEs are typical of target-independent / free toxin-related toxicities. Therefore, compared to belentuximab vedoltin, belentuximab tub-tag ADCs are expected to have a more favorable clinical safety profile with fewer target-independent / free MMA-related toxicities. Overall, the data presented suggest a better safety profile for belentuximab tub-tag ADCs compared to belentuximab vedoltin in rats.Therefore, clinically, belenutoxic tub-tag ADCs are unlikely to cause the specific dose-limiting toxicities of belenutoxic vedotin, including anemia, neutropenia, and hyperglycemia. The lack of target-independent / free toxin-related toxicities associated with belenutoxic vedotin in belenutoxic ADCs also offsets characteristic changes such as peripheral neuropathy seen in many ADCs containing MMAEs, including belenutoxic vedotin. Example 7: Repeated-dose toxicity study of belenutoxic tub-tag MMAEs in stone crab macaques.

[0337] Next, the toxicity of belenutoxic acid Tub-tag MMAE (Bren. LC-2) was evaluated in lithocrad macaques. The aim of this study was to evaluate the toxicity and toxicokinetics (TK) of belenutoxic acid Tub-tag MMAE (Bren. LC-2) in lithocrad macaques. Belenutoxic acid Tub-tag ADC was administered at 6, 12, and 15 mg / kg (6 mg / kg Q3Wx4, 12 and 15 mg / kg Q3Wx2). Clinical status (including body weight, food consumption, and clinical observation), clinicopathology, and immunophenotype (T, B, and NK cells) were assessed throughout the study, and samples were taken frequently to determine TK and anti-drug antibodies. All animals underwent full necropsy and detailed macroscopic observation. Belenutoxic acid Tub-tag MMAE (Bren. LC-2) was well tolerated clinically at 6, 12, and 15 mg / kg and had no effect on body weight. According to FDA PharmTox review, rhesus monkeys were poorly tolerated at 6 mg / kg (MTD 3 mg / kg), dying within 11 to 15 days after the first dose and undergoing early euthanasia. In contrast, no mortality or macroscopic or serious minor findings were observed with belenutoxicillin Tub-tag MMAE (Bren. LC-2) in this study. Decreases in erythrocytes, hemoglobin, and neutrophils were noted with belenutoxicillin Tub-tag MMAE (Bren. LC-2) at 12 and 15 mg / kg, which are relatively consistent with those observed with Adcetris at 3 mg / kg (historical data). Furthermore, as shown in Figure 19, belenutoxicillin Tub-tag MMAE (Bren. LC-2) exhibited high stability in toxicological analysis.

[0338] To analyze the changes in ADC content in the serum of rhesus macaques over time, an ELISA-based assay has been developed to detect antibody and intact ADC content (by measuring the effective load linked to the antibody; the same ELISA assay setup was used in pharmacokinetic and repeated-dose toxicity studies in rats, except that this assay has been further validated for use in rhesus macaques). The total antibody assay provides useful information about antibody clearance, and the difference detected compared to the intact ADC assay can explain the loss of antibody effective load. The high overlap of the intact ADC and total monoclonal antibody curves in Figure 19 shows that belentoxigenic Tub-tag MMAE has minimal effective load loss and high ADC stability.

[0339] Figure 19 shows the toxicokinetic analysis of belenutoxicillin Tub-tag MMAE (i.e., Bren. LC-2) administered at doses of 12 and 15 mg / kg in Lithocarpus macaques. The total amount of monoclonal antibodies and intact ADCs was assessed by ELISA. The high overlap of the intact ADC and total monoclonal antibody curves demonstrates the high stability of belenutoxicillin Tub-tag MMAE at both dose concentrations. During the repeated-dose study of belenutoxicillin Tub-tag MMAE (Bren. LC-2), the body weight and different blood values, namely the concentrations of lymphocytes, neutrophils, and leukocytes, were monitored in Lithocarpus macaques. Figure 20 shows the selected values ​​of body weight and different blood values ​​collected in the repeated-dose study of belenutoxicillin Tub-tag MMAE (Bren. LC-2) in Lithocarpus macaques. More specifically, the upper left panel of Figure 20 shows the change in body weight over time. The upper right panel of Figure 20 shows the change in lymphocyte concentration over time. The lower left panel of Figure 20 shows the change in neutrophil concentration over time. The lower right panel of Figure 20 shows the change in white blood cell concentration over time. In each case, data are shown as the mean and error for repeated administration of 6, 12, and 15 mg / kg belentoxigenic tumouretine (Bren. LC-2) to two female animals in three groups, and compared with the results of a toxicity study reported by the FDA in "Clinical Pharmacology and Biopharmaceutics Review(s)" (application number: 125388Orig1S00) (available at https: / / www.accessdata.fda.gov / drugsatfda_docs / nda / 2011 / 125388orig1s000clinpharmr.pdf). Body weight is in kilograms, lymphocytes are in billion per liter, neutrophils are in billion per liter, and white blood cells are in billion per liter. One of the dose-limiting toxicities of belentuxivdocin in clinical practice is neutropenia. The data set shown in the lower left panel of Figure 20 demonstrates that, compared directly to belentuxivdocin, even administration of two times (6 mg / kg), four times (12 mg / kg), and five times (15 mg / kg) the dose of belentuxivdocin (3 mg / kg) did not preferentially result in more neutropenia with belentuxivdocin. Additionally, weight gain was observed (upper left panel of Figure 20). No significant changes in lymphocyte or white blood cell concentrations were detected (upper right and lower right panels of Figure 20). The combination of observed neutrophil concentrations, weight gain in all animals throughout the study, and no significant changes in white blood cell and lymphocyte concentrations clearly demonstrates that the ADC described in this paper is well-tolerated, exceeding the tolerability of belentuxivdocin.Therefore, the data clearly show that it is well-tolerated in clinical practice, exceeding the tolerability of belentoxividodin.

[0340] In summary, the results presented demonstrate that the berentoxigenic Tub-tag ADC is characterized by unexpectedly high stability in vitro and in vivo, which translates into excellent in vivo efficacy and toxicological properties. [Simplified Explanation of the Diagram]

[0023] The invention will be better understood by referring to the embodiments when considered in conjunction with non-limiting examples and the accompanying drawings, wherein:

[0024] Figure 1 shows analytical particle size screening (SEC) chromatography after protein A chromatography (PAC). Figure 1A shows the SEC of unmodified belentoxicin, and Figures 1B-D show the SEC of the Tub-tag variant of belentoxicin. The monoclonal antibody (mAb) is highly monomeric after expression and purification.

[0025] Figure 2 shows the analysis of belentoxicin (black) and its Tub-tag analogues (abbreviated as "Bren."), denoted as Bren. LC-Tub (gray, dashed line), Bren. HC-Tub (gray), and Bren. LCHC-Tub (black, dashed line). Figure 2A is a hydrophilic interaction chromatography (HIC) chromatogram, showing the normalized absorption spectrum recorded at 220 nm. Retention time is a measure of hydrophobicity. Figure 2B shows the differential scanning fluorescence (DSF) method used to determine the melting point (Tm).

[0026] Figure 3 illustrates the analysis of belentoxib Tub-tag ADCs via analytical SEC and HIC. Each belentoxib variant (abbreviated as "Bren.") was expressed, purified with PAC, and conjugated with payload 2 to produce DAR 2, and conjugated with either payload 2 or payload 3 to produce DAR 4 ADCs, where Figures 3A and D show Bren.HC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren.HC-2"), Figures 3B and E show Bren.LC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren.LC-2"), and Figures 3C and F show Bren.LCHC-vc-PAB-MMAE DAR4 (also referred to herein as "Bren.LCHC-2"). After final polishing with HIC and buffer exchange, the Tub-tag ADCs were analyzed based on aggregate content and DAR homogeneity. All Tub-tag ADCs contain very low HMWS (< 1%) and exhibit excellent DAR homogeneity.

[0027] Figure 4 shows the analysis of berentoxicin Tub-ta...

Claims

1. An antibody-drug conjugate (ADC) comprising: (a) brentuximab, wherein brentuximab contains a microtubule tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, heavy chain, or all of the heavy chain and the light chain of the brentuximab; and (b) at least one pharmaceutical moiety; wherein the pharmaceutical moiety is conjugated to each of the non-natural amino acids via a linker.

2. The ADC of claim 1, wherein the heavy chain of belentoxidine has an amino acid sequence comprising or consisting of SEQ ID NO: 1, or having at least 95% sequence identity with SEQ ID NO: 1, wherein optionally the last residue K at position 447 of the heavy chain may be omitted; and / or wherein the light chain of belentoxidine has an amino acid sequence comprising or consisting of SEQ ID NO: 2, or having at least 95% sequence identity with SEQ ID NO:

2.

3. The ADC of claim 2, wherein belentoxidine consists of a heavy chain and a light chain, wherein the heavy chain consists of the amino acid sequence of SEQ ID NO: 1, wherein optionally the last residue K at position 447 of the heavy chain may be omitted, and the light chain consists of the amino acid sequence of SEQ ID NO:

2.

4. For ADCs requested in items 1, 2, or 3, the drug portion is selected from the group consisting of: camptothecin, maytansinoid, calicheamycin, duocarmycin, tubulolysin, amatoxin, dolastatin, and auristatin, pyrrolobenzodiazepine dimer, indolinobenzodiazepine dimer, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTAC, kinase inhibitors, MEK inhibitors, and KSP inhibitors.

5. As in request item 4, the ADC, wherein the drug component is MMAE.

6. The ADC of claim 1, 2 or 3, wherein the recognition sequence of the tubulin tyrosine ligase has at least the amino acid sequence X1X2X3X4 (SEQ ID NO: 3), wherein X1 and X2 are any amino acids, X3 is E, D or C and X4 is E.

7. The ADC of request item 6, wherein X2 is G, S, A, V or F, and / or wherein X1 is E, D, A, K or P.

8. The ADC of claim 7, wherein the identification sequence is EGEE (SEQ ID No. 4).

9. The ADC of claim 7, wherein the identification sequence is VDSVEGEGEEEGEE (SEQ ID No. 5), SVEGEGEEEGEE (SEQ ID No. 6), SADGEDEGEE (SEQ ID No. 7), SVEAEAEEGEE (SEQ ID No. 8), SYEDEDEGEE (SEQ ID No. 9), or SFEEENEGEE (SEQ ID No. 10).

10. An ADC as claimed in claim 1, 2 or 3, wherein the non-natural amino acid is a 2-substituted, 3-substituted or 4-substituted tyrosine, or a tyrosine substituted at the benzyl position.

11. The ADC of claim 10, wherein the 3-substituted or 4-substituted tyrosine is 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-methoxytyrosine, 3-acetyrosine, or 4-aminophenylalanine.

12. The ADC of claim 11, wherein the 3-substituted or 4-substituted tyrosine is 3-methoxytyrosine.

13. An ADC as requested in items 1, 2 or 3, wherein the connector is detachable.

14. The ADC of claim 13, wherein the linker is protease-cleavable.

15. The ADC of claim 14, wherein the protease is a cathepsin.

16. An ADC as requested in item 1, 2 or 3, wherein the linker comprises a valine-citrulline portion.

17. An ADC as claimed in claim 1, 2 or 3, wherein the linker comprises a hydroxylamine group and the non-natural amino acid comprises a formyl group located ortho to a hydroxyl group in an aromatic ring, and wherein, after conjugation, the hydroxylamine group of the linker forms an oxime with the formyl group of the non-natural amino acid.

18. An ADC such as requested in items 1, 2 or 3, wherein belentoxicin is conjugated with two, four, six or eight drug portions.

19. An ADC as requested in claim 18, wherein belentoxicin is conjugated with two or four drug components.

20. An ADC as requested in claim 19, wherein belentoxicin is partially conjugated with both drugs.

21. An ADC as claimed in claim 1, 2 or 3, wherein the linker has a structure as shown in structure 1 before being conjugated with the non-natural amino acid: , wherein R is one or more pharmaceutical moieties that optionally are conjugated with the hydroxylamine of structure 1 via one or more cleavage sites.

22. The ADC of claim 21, wherein the hydroxylamine of structure 1 is conjugated with the non-natural amino acid.

23. An ADC as claimed in claim 1, 2, or 3, wherein the linker has a structure as shown in structure 2 or 3 before conjugation with the non-natural amino acid: , wherein Z is selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; wherein D is one or more pharmaceutical moieties; and wherein Y is a cleavage site.

24. The ADC of claim 23, wherein the hydroxylamine of structure 2 or structure 3 is conjugated with the non-natural amino acid.

25. An ADC as claimed in claim 1, 2 or 3, wherein the linker has a structure as shown in structure 4 or 5 before being conjugated with the non-natural amino acid, wherein D is the pharmaceutical part: .

26. The ADC of claim 25, wherein the non-natural amino acid is 3-methoxytyrosine, and the hydroxylamine group of the linker forms an oxime with the 3-methoxy group of the non-natural amino acid.

27. The ADC of request item 26, wherein the drug component is MMAE.

28. An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1, wherein optionally the last residue K at position 447 of the heavy chain is omitted; and (b) the C-terminus of the recognition sequence of each light chain is bound via a amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

29. An antibody-drug conjugate (ADC) comprising: (a) belentoxigenin, wherein belentoxigenin contains a microtubule-tyrosine ligase recognition sequence at the C-terminus of each light chain; each light chain, including the recognition sequence, has SEQ ID NO: 11; and each heavy chain of belentoxigenin has SEQ ID NO: 1, wherein optionally the last residue K at position 447 of the heavy chain is omitted; and (b) the C-terminus of the recognition sequence of each light chain is bound via a amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

30. An antibody-drug conjugate (ADC) comprising: (a) an anti-CD30 IgG antibody consisting of two light chains and two heavy chains, wherein the heavy chains consist of the amino acid sequence shown in SEQ ID NO: 1, wherein optionally the last residue K at position 447 of the heavy chain is omitted, and wherein the light chains consist of the amino acid sequence shown in SEQ ID NO: 11; and (b) the C-terminus of each light chain is bound via an amide bond to a group having the following structure: , wherein the wavy line indicates the connection to the C-terminus of the recognition sequence of each light chain.

31. A method of manufacturing an ADC as defined in any one of claims 1 to 30, comprising: (a) introducing or adding a microtubule tyrosine ligase recognition sequence to the C-terminus of a light chain, a heavy chain, or both a light chain and a heavy chain of belentoxicin; (b) contacting the belentoxicin obtained in step (a) in the presence of microtubule tyrosine ligase and a non-natural amino acid, under conditions suitable for microtubule tyrosine ligase to link the belentoxicin to the non-natural amino acid; and (c) conjugating an optional cleavable linker comprising a pharmaceutical moiety to the linked belentoxicin obtained in step (b).

32. A pharmaceutical composition comprising the ADC of any one of claims 1 to 30.

33. Use of an ADC as claimed in any one of claims 1 to 30, for the preparation of a medicament for treating diseases associated with overexpression of CD30.

34. For the purposes of claim 33, wherein the disease is selected from the group consisting of: lymphoma, non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemia, acute lymphoblastic leukemia (ALL) and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE). ); or the disease is selected from the following groups: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma.

35. As claimed in claim 34, wherein the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL); or wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL).

36. Use of a pharmaceutical composition as claimed in claim 32, for the preparation of a medicament for treating diseases associated with overexpression of CD30.

37. For the purposes of claim 36, wherein the disease is selected from the group consisting of: lymphoma, non-Hodgkin's lymphoma (NHL), degenerative large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma, and enteropathy-associated T-cell lymphoma (EATL); leukemia, acute lymphoblastic leukemia (ALL) and mast cell leukemia; germ cell carcinoma; graft-versus-host disease (GvHD); and lupus, particularly systemic lupus erythematosus (SLE). ); or the disease is selected from the following groups: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enterolesion-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal γ / δ-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma.

38. As claimed in claim 37, wherein the disease is Hodgkin's lymphoma (HL) or degenerative large cell lymphoma (ALCL); or wherein the disease is peripheral T-cell lymphoma (PTCL), including degenerative large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous degenerative large cell lymphoma (pcALCL).

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