Antibody drug conjugates having a linker comprising a hydrophilic group

Abstract

Provided herein are linkers, linker-drug groups, and antibody-drug conjugates comprising hydrophilic groups.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 850,094, filed May 20, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention provides a linker for improving the solubility of antibody-drug conjugates (ADCs) comprising one or more hydrophobic drug compounds. Background Technology

[0004] One aspect of antibody-drug conjugate (ADC) design is the design of chemical linkers that connect the drug portion to the target portion. Typically, ADCs use hydrophobic drug portions, but when such drug portions are combined with relatively hydrophobic linkers, solubility issues arise, which can affect the biocompatibility and drug efficacy of the ADC.

[0005] Reportedly, various linker strategies have been developed to address these challenges, particularly hydrophilic linkers incorporating polyethylene glycol (see R. Plyon, T.D. Bovee, S.O. Doronina, P.J. Burke, J.H. Hunter, H.D. N. E. LaFord, M. Jonas, M.E. Anderson, J.R. Stetter, P.D. Senter, Nat. Biotechnol. [Nature Biotechnology], 2015, 33, 733-735 and WO 2015057699), linkers incorporating sulfonates (R.Zhao, S.D. Wilhelm, C. Audette, G. Jones, B.A. Leece, C.A. C. A. L. Zhar, V.S. Goldmacher, R. Singh, Y. Kovtun, W.C. Widdison, J.M. Lambert, RV. J. Chari, J. Med. Chem. [Journal of Medicinal Chemistry], 2011, 54, 3606-3623), and linkers with a carbohydrate backbone (F.S. Kholm, H.). A.Vilkman, V. The design of J. Helin, J. Saarinen, T. Satomaa, ChemMedChem. [Chemical Pharmaceutical Chemistry], 2016, 11(22):2501-2505). However, there is still a need for antibody-drug conjugates that allow for targeted delivery of hydrophobic drugs with improved pharmacokinetic and pharmacodynamic properties. Summary of the Invention

[0006] This invention provides a connector for use in improving the solubility of connector-drug conjugates, wherein such conjugates comprise one or more hydrophobic drug compounds, and wherein the connector comprises one or more hydrophilic groups. Various embodiments of the invention are described herein.

[0007] The present invention further provides a linker for use in improving the solubility of antibody-drug conjugates (ADCs), wherein the ADC comprises one or more hydrophobic drug compounds, and wherein the linker comprises one or more hydrophilic groups. Various embodiments of the invention are described herein.

[0008] In one embodiment, this document discloses a connector comprising one or more suicide groups, wherein each of the one or more suicide groups is replaced by one or more hydrophilic portions.

[0009] In one embodiment, this document discloses a connector-drug group, wherein the connector comprises one or more suicide groups coupled to a drug, and wherein each of the one or more suicide groups is replaced by one or more hydrophilic portions.

[0010] In one embodiment, this document discloses an antibody-drug conjugate comprising one or more linker-drug groups, wherein the linker comprises one or more suicide groups coupled to a drug, and wherein each of the one or more suicide groups is replaced by one or more hydrophilic moieties.

[0011] In one embodiment, it is a linker-drug group having formula (I), or a pharmaceutically acceptable salt thereof:

[0012]

[0013] in:

[0014] R 1 It is a reactive group;

[0015] L1 is a bridging spacer;

[0016] Lp is a divalent peptide spacer;

[0017] G-L2-A is a suicide spacer;

[0018] R 2 It is the hydrophilic part;

[0019] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0020] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0021] L3 is the spacer sub-part;

[0022] and

[0023] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0024] In embodiments having a linker-drug group of formula (I), it is a linker-drug group of formula (II), or a pharmaceutically acceptable salt thereof:

[0025]

[0026] in:

[0027] R 1 It is a reactive group;

[0028] L1 is a bridging spacer;

[0029] Lp is a divalent peptide linker containing one to four amino acid residues;

[0030] R 2 It is the hydrophilic part;

[0031] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0032] L3 is the spacer sub-part;

[0033] and

[0034] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0035] In one embodiment, it is an antibody-drug conjugate having formula (III):

[0036]

[0037] in:

[0038] Ab is an antibody or a fragment thereof;

[0039] R 100 It is a coupling group;

[0040] L1 is a bridging spacer;

[0041] Lp is a divalent peptide linker;

[0042] G-L2-A is a suicide spacer;

[0043] R 2 It is the hydrophilic part;

[0044] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0045] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0046] L3 is the spacer sub-part;

[0047] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0048] and

[0049] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0050] In the examples of antibody drug conjugates having formula (III), there are antibody drug conjugates having formula (IV):

[0051]

[0052] in:

[0053] Ab is an antibody or a fragment thereof;

[0054] R 100 It is a coupling group;

[0055] L1 is a bridging spacer;

[0056] Lp is a divalent peptide linker containing one to four amino acid residues;

[0057] R 2 It is the hydrophilic part;

[0058] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0059] L3 is the spacer sub-part;

[0060] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0061] and

[0062] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0063] Another aspect of the invention is a connector having a structure of formula (V).

[0064]

[0065] in

[0066] L1 is a bridging spacer;

[0067] Lp is a divalent peptide spacer;

[0068] G-L2-A is a suicide spacer;

[0069] R 2 It is the hydrophilic part;

[0070] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0071] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a)2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the ** of A indicates the attachment point with L2.

[0072] and

[0073] L3 is the spacer sub-part.

[0074] In an embodiment of the connector having type (V), there is a connector having the structure of type (VI).

[0075]

[0076] in

[0077] L1 is a bridging spacer;

[0078] Lp is a divalent peptide spacer;

[0079] R 2 It is the hydrophilic part;

[0080] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl, or C3-C8 cycloalkyl.

[0081] and

[0082] L3 is the spacer sub-part.

[0083] The linkers described herein contain a hydrophilic moiety, which contributes to the overall hydrophilicity of antibody-drug conjugates (ADCs) and improves the water solubility of said ADCs. The linkers also unexpectedly reduce ADC aggregation and improve the pharmacokinetic and pharmacodynamic properties of ADCs. Furthermore, the hydrophilic linkers described herein allow for improved water solubility of the linker-drug group, thereby allowing for improved antibody conjugation to the linker-drug group, which improves the purification and overall synthetic yield of ADCs, particularly those containing a hydrophobic drug moiety. Attached Figure Description

[0084] Figure 1 Line graph of cell viability of antibody-drug conjugates titrated in selected cell lines (A: HT-29PCAD+; B: FaDu; C: HCC70; D: HT-29; and E: HCC1954).

[0085] Figure 2 A: Line graphs of caspase-3 / 7 activity of antibody-drug conjugates titrated in HCC1954 cell line after 24 hours and B: after 48 hours.

[0086] Figure 3 Efficacy and tolerability of PCAD-ADC and huIgG1 isotype-matched control ADC in the HCC70 human TNBC xenograft model in SCID-beige female mice. A) Antitumor response; B) and C) Changes in body weight compared to body weight at the start of treatment. Data are presented as mean ± SEM. *p<0.05, compared to the untreated group at day 20 (one-way ANOVA and post-hoc Dunnett test). Detailed Implementation

[0087] This document describes various enumerated embodiments of the invention. It should be understood that the features specified in each embodiment can be combined with other specified features to provide further embodiments of the invention.

[0088] In the entirety of this application, if there are any discrepancies between the specification text (e.g., Table 3) and the sequence list, the specification text shall prevail.

[0089] definition

[0090] As used herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and said group is not unsaturated. As used herein, the term "C1-C6 alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, said group is not unsaturated, has one to six carbon atoms, and is attached to the rest of the molecule by single bonds. Non-limiting examples of "C1-C6 alkyl" groups include methyl (C1 alkyl), ethyl (C2 alkyl), 1-methylethyl (C3 alkyl), n-propyl (C3 alkyl), isopropyl (C3 alkyl), n-butyl (C4 alkyl), isobutyl (C4 alkyl), sec-butyl (C4 alkyl), tert-butyl (C4 alkyl), n-pentyl (C5 alkyl), isopentyl (C5 alkyl), neopentyl (C5 alkyl), and hexyl (C6 alkyl).

[0091] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, wherein the group contains at least one double bond. As used herein, the term "C2-C" refers to... e"Alkenyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one double bond, having two to six carbon atoms attached to the rest of the molecule by single bonds. Non-limiting examples of "C2-C6 alkenyl" groups include vinyl (C2 alkenyl), propenyl (C3 alkenyl), butenyl (C4 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), hexenyl (C6 alkenyl), and hexenyl (C6 alkenyl). Hex-3-enyl (C6-enyl), hex-1,4-dienyl (C6-enyl), hex-1,5-dienyl (C6-enyl), and hex-2,4-dienyl (C6-enyl). As used herein, the term "C2-C3-enyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one double bond, having two to three carbon atoms attached to the remainder of the molecule by single bonds. Non-limiting examples of "C2-C3-enyl" groups include vinyl (C2-enyl) and propenyl (C3-enyl).

[0092] As used herein, the term "alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and wherein the group is not unsaturated. As used herein, the term "C1-C6 alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, wherein the group is not unsaturated, and has one to six carbon atoms. Non-limiting examples of "C1-C6 alkylene" groups include methylene (C1 alkylene), ethylene (C2 alkylene), 1-methylethylene (C3 alkylene), n-propylene (C3 alkylene), isopropylene (C3 alkylene), n-butylene (C4 alkylene), isobutylene (C4 alkylene), secondary butylene (C4 alkylene), tert-butylene (C4 alkylene), n-pentylene (C5 alkylene), isopentylene (C5 alkylene), neopentylene (C5 alkylene), and hexylene (C6 alkylene).

[0093] As used herein, the term "alkenyl" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and said group contains at least one double bond. As used herein, the term "C2-C6 alkenyl" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, said group contains at least one double bond, and has two to six carbon atoms. Non-limiting examples of “C2-C6 alkenyl” groups include vinylene (C2 alkenyl), propenyl-1-alkenyl (C3 alkenyl), butenyl-1-alkenyl (C4 alkenyl), pentylene-1-alkenyl (C5 alkenyl), pentylene-4-alkenyl (C5 alkenyl), pentylene-1,4-dienyl (C5 alkenyl), hexene-1-alkenyl (C6 alkenyl), hexene-2-alkenyl (C6 alkenyl), hexene-3-alkenyl (C6 alkenyl), hexene-1,4-dienyl (C6 alkenyl), hexene-1,5-dienyl (C6 alkenyl), and hexene-2,4-dienyl (C6 alkenyl). As used herein, the term “C2-C6 alkenyl” refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, the group containing at least one double bond, and having two to three carbon atoms. Non-limiting examples of “C2-C3 imenyl” groups include vinylidene (C2 imenyl) and propylene-1-enyl (C3 imenyl).

[0094] As used herein, the term "cycloalkyl" or "C3-C8 cycloalkyl" refers to a saturated, monocyclic, fused bicyclic, fused tricyclic, or bridged polycyclic ring system. Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane, and adamantyl. Non-limiting examples of monocyclic C3-C8 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

[0095] As used herein, the terms "polyethylene glycol" or "PEG" refer to a linear, branched, or star-shaped conformation consisting of (OCH2CH2) groups. In some embodiments, the polyethylene or PEG groups are -(OCH2CH2). t *-, where t is 4-40, and where "-" indicates the end of the suicide spacer and "*-" indicates the attachment point to the terminal group R', where R' is OH, OCH3, or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -(CH2CH2O). t *-, where t is 4-40, and where “-” indicates the end of the suicide spacer and “*-” indicates the attachment point to the terminal group R”, where R” is H, CH3 or CH2CH2C(=O)OH.

[0096] As used herein, the term "polyethylene glycol" refers to polyethylene glycol composed of (O(CH2)2) m ) t The groups form linear chains, branched chains, or star-shaped conformations. In some embodiments, the polyethylene or PEG groups are -(O(CH2)). m ) t *-, where m is 1-10, t is 4-40, and where "-" indicates the end of the suicide spacer and "*-" indicates the attachment point to the terminal group R', where R' is OH, OCH3, or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -((CH2) m O) t *-, where m is 1-10, t is 4-40, and where “-” indicates the end of the suicide spacer and “*-” indicates the attachment point to the terminal group R”, where R” is H, CH3 or CH2CH2C(=O)OH.

[0097] As used herein, the terms "pharmaceutical moiety," "D," or "pharmaceutical" refer to any compound having the desired biological activity and reactive functional group, which can be used to incorporate the pharmaceutical into the connector-pharmaceutical group of the present invention. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing diseases in humans or other animals. The reactive functional group forms a bond with "A" in compounds having formulas (I) and (II) and in conjugates having formulas (III) and (IV). In some embodiments, the pharmaceutical moiety has a nitrogen atom that can form a bond with "A." In other embodiments, the pharmaceutical moiety has a hydroxyl group that can form a bond with "A." In other embodiments, the pharmaceutical moiety has a carboxylic acid that can form a bond with "A." In other embodiments, the pharmaceutical moiety has a carbonyl group that can form a bond with "A." In still other embodiments, the pharmaceutical moiety has a thiol group that can form a bond with "A."

[0098] If the required reactive functional group is present, the terms "drug part," "D," or "drug" further refer to a chemical or any supplement thereof identified as a drug in the official United States Pharmacopeia, the official Homeopathic Pharmacopeia of the United States, or the official National Formulary. Exemplary drugs are provided in the Physician's Desk Reference (PDR) and the Orange Book maintained by the U.S. Food and Drug Administration (FDA).

[0099] In one embodiment, the pharmaceutical portion (D) may be a cytotoxic drug, a cell growth inhibitor, or an immunosuppressive drug. Such cytotoxic or immunosuppressive drugs include, for example, anti-tubulin agents, tubulin inhibitors, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolate agents, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of such cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycins, etoposide, maytansine and maytansinoid alkaloids, taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBD), indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vinca alkaloids.

[0100] In embodiments where the pharmaceutical portion is hydrophobic, the effects of the present invention will be more pronounced. Therefore, the pharmaceutical portion of the present invention is preferably hydrophobic, having a SlogP value of 1.5 or greater, 2.0 or greater, or 2.5 or greater. In some aspects, the pharmaceutical portion used in the present invention has a SlogP value of (a) from about 1.5, about 2, or 2.5 to about 7, (b) from about 1.5, about 2, or 2.5 to about 6, (c) from about 1.5, about 2, or about 2.5 to about 5, (d) from about 1.5, about 2, or 2.5 to about 4, or (e) from about 1.5, about 2, or about 2.5 to about 3.

[0101] Hydrophobicity can be measured using SlogP. SlogP is defined as the logarithm of the octanol / water partition coefficient (including implicit hydrogens) and can be measured using MOE from the Chemical Calculations Group. TMThe program calculates (using Wildman, 25 S.A., Crippen, GM; Prediction of Physiochemical Parameters by Atomic Contributions; J. Chern.lnf Comput. Sci. 39 No. 5 (1999) 868-873) to calculate the SlogP value).

[0102] As used herein, the term "reactive group" is a functional group capable of forming a covalent bond with a functional group of an antibody or antibody fragment. Non-limiting examples of such functional groups include the reactive groups provided in Table 1 herein.

[0103] As used herein, the term "coupling group" refers to a divalent moiety that links a bridging spacer to an antibody or a fragment thereof. The coupling group is a divalent moiety formed by a reaction between a reactive group and a functional group of the antibody or a fragment thereof. Non-limiting examples of such divalent moieties include the divalent chemical moieties given in Tables 1 and 2 provided herein.

[0104] As used herein, the term "bridging spacer" refers to one or more linker components covalently attached together to form a divalent moiety that links the divalent peptide spacer to a reactive group or to a coupling group. In some embodiments, the "bridging spacer" comprises a carboxyl group attached to the N-terminus of the divalent peptide spacer via an amide bond.

[0105] As used herein, the term "spacer portion" refers to one or more connector components that are covalently attached together to form a portion that connects a suicide spacer to a hydrophilic portion.

[0106] As used herein, the term "divalent peptide spacer" refers to a divalent linker comprising one or more amino acid residues covalently attached together to form a portion that connects a bridging spacer to a suicide spacer. The one or more amino acid residues may be residues selected from the following amino acids: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), pentaneous (Nva), ortholeucine (Nle), selenocysteine ​​(Sec), pyrrolidone (Pyl), homoserine, homocysteine, and demethylpyrrolidone.

[0107] In some embodiments, the "divalent peptide spacer" is a combination of two to four amino acid residues, wherein each residue is independently selected from residues selected from the following amino acids: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), and proline (Pro). Glutamine (Gln), Arginine (Arg), Serine (Ser), Threonine (Thr), Valine (Val), Tryptophan (Trp), Tyrosine (Tyr), Citrulline (Cit), Pentylene (Nva), Leucine (Nle), Selenocysteine ​​(Sec), Pyrrolidone (Pyl), Homoserine, Homocysteine, and Demethylpyrrolidone, for example -ValCit*; -CitVal*; -AlaAla*; -AlaCit*; -CitAla*; - AsnCit*;-CitAsn*;-CitCit*;-ValGlu*;-GluVal*;-SerCit*;-CitSer*;-LysCit*;-CitLys*;-AspCit*;-Ci tAsp*;-AlaVal*;-ValAla*;-PheAla*;-AlaPhe*;-PheLys*;-LysPhe*;-ValLys*;-LysVal*;-AlaLys*;-LysAl a*;-PheCit*;-CitPhe*;-LeuCit*;-CitLeu*;-IleCit*;-CitIle*;-PheArg*;-ArgPhe*;-CitTrp*;-TrpCit* ;-PhePheLys*;-LysPhePhe*;-DPhePheLys*;-DLysPhePhe*;-GlyPheLys*;-LysPheGly*;-GlyPheLeuGly-[SEQ [SEQ ID NO:160]; -GlyLeuPheGly-[SEQ ID NO:161]; -AlaLeuAlaLeu-[SEQ ID NO:162]; -GlyGlyGly*; -GlyGlyGlyGly-[SEQ ID NO:163]; -GlyPheValGly-[SEQ ID NO:164]; and -GlyValPheGly-[SEQ ID NO:165], wherein "-" indicates the attachment point to the bridging spacer and "*" indicates the attachment point to the suicide spacer.

[0108] As used herein, the term "joint component" refers to the following:

[0109] a) Alkyl group: -(CH2) n -, which can be linear or branched (where in this case, n is 1-18);

[0110] b) Alkenyl group;

[0111] c) Alynyl group;

[0112] d) Alkenyl groups;

[0113] e) Acynyl group;

[0114] f) Ethylene glycol unit: -OCH2CH2 or -CH2CH2O;

[0115] g) Polyethylene glycol unit: (-CH2CH2O-) x (where x is 2-20 in this case);

[0116] h)-O;

[0117] i)-S;

[0118] j) Carbonyl group: -C (=O);

[0119] k) Ester: -C(=O)-O- or -OC(=O);

[0120] l) Carbonate: -OC(=O)O;

[0121] m)Amine: -NH;

[0122] n) Tertiary amines

[0123] o) Amides: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C 1-6 alkyl);

[0124] p) Carbamate: -OC(=O)NH- or -NHC(=O)O;

[0125] q) Urea: -NHC(=O)NH;

[0126] r) Sulfonamides: -S(O)2NH- or -NHS(O)2;

[0127] s) Ethers: -CH2O- or -OCH2;

[0128] t) an alkylene group substituted with one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0129] u) an alkenyl group substituted by one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0130] v) An alkynyl group substituted by one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0131] w)C1-C 10 Alkylene, wherein one or more methylene groups are partially replaced by one or more -S-, -NH- or -O- groups;

[0132] x) A ring system having two available attachment sites, for example selected from the following divalent rings: phenyl (including 1,2-, 1,3- and 1,4-disubstituted phenyl), C5-C6 heteroaryl, C3-C8 cycloalkyl (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and 1,4-disubstituted cyclohexyl), and C4-C8 heterocycloalkyl;

[0133] y) is selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), pentaneous (Nva), ortholeucine (Nle), selenocysteine ​​(Sec), pyrrolidone (Pyl), homoserine, homocysteine, and demethylpyrrolidone;

[0134] A combination of two or more amino acid residues, wherein each residue is independently selected from residues of the following amino acids: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), pentaneous (Nva), noreleucine (Nle), selenocysteine ​​(Sec), pyrrolidone (Pyl), homoserine, homocysteine Acids and demethylpyrrolidones, such as Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit;

[0135] as well as

[0136] z) A suicide spacer, wherein the suicide spacer comprises one or more protecting (triggering) groups, the protecting (triggering) groups being susceptible to acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.

[0137] Non-limiting examples of such suicide spacers include:

[0138]

[0139] in:

[0140] PG is a protecting (triggering) group;

[0141] X a It is O, NH or S;

[0142] X b It is O, NH, NCH3, or S;

[0143] X c It is O or NH;

[0144] Y a It is CH2, CH2O, or CH2NH;

[0145] Y b It is CH2, O, or NH;

[0146] Y c It is a bond, CH2, O or NH, and

[0147] LG is a leaving group, such as the pharmaceutical portion (D) of the linker-pharmaceutical group of the present invention.

[0148] Further non-limiting examples of such suicide spacers are described in Angew. Chem. Int. Ed. [Applied Chemistry International Edition] 2015, 54, 7492-7509.

[0149] Additionally, the linker component can be a chemical motif that is readily formed through a reaction between two reactive groups. Non-limiting examples of such chemical motifs are given in Table 1.

[0150] Table 1

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Wherein: R in Table 1 32 It is H, C 1-4 Alkyl, phenyl, pyrimidine, or pyridine; R in Table 1 35 It is H, C 1-6 Alkyl, phenyl, or C substituted with 1 to 3 -OH groups 1-4 Alkyl; each R in Table 1 7 Independently selected from H and C 1-6 Alkyl, fluorine, benzyloxy group substituted with -C(=O)OH, benzyl group substituted with -C(=O)OH, C group substituted with -C(=O)OH 1-4 Alkoxy groups and C groups substituted with -C(=O)OH 1-4 Alkyl; R in Table 1 37Independently selected from H, phenyl, and pyridine; q in Table 1 is 0, 1, 2, or 3; R in Table 1 8 or R 13 It is H or methyl; and R in Table 1 9 or R 14 It is H, -CH3, or phenyl; and R in Table 1 is H or a suitable substituent, such as alkyl.

[0158] In addition, the connector component can be one of the groups given in Table 2 below.

[0159] Table 2

[0160]

[0161]

[0162]

[0163]

[0164] As used in this article, wavy lines are used when showing partial structures of compounds. Indicates the attachment points between the described partial structure and the rest of the molecule.

[0165] As used herein, the term "suicide spacer" refers to a portion containing one or more trigger groups (TGs) that are activated by acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage, and upon activation, the protecting group is removed, resulting in a cascade of decomposition reactions leading to the temporary sequential release of leaving groups. Such cascade reactions can be, but are not limited to, 1,4-, 1,6-, or 1,8-elimination reactions.

[0166] Non-limiting examples of such suicide spacers include:

[0167] These groups can be optionally substituted, and

[0168] in:

[0169] TG is a trigger group;

[0170] X a It is O, NH or S;

[0171] X b It is O, NH, NCH3, or S;

[0172] X c It is O or NH;

[0173] Y a It is CH2, CH2O, or CH2NH;

[0174] Y b It is CH2, O, or NH;

[0175] Y c It is a bond, CH2, O or NH, and

[0176] LG is a leaving group, such as the pharmaceutical portion (D) of the linker-pharmaceutical group of the present invention.

[0177] Further non-limiting examples of such suicide spacers are described in Angew. Chem. Int. Ed. [Applied Chemistry International Edition] 2015, 54, 7492-7509.

[0178] In some embodiments, the suicide spacer is a portion having the following structure:

[0179] Lp is an enzymatically cleavable divalent peptide spacer, and A, D, L3, and R... 2 As defined in this article.

[0180] In a preferred embodiment, the suicide spacer is a portion having the following structure:

[0181] Lp is an enzymatically cleavable divalent peptide spacer, and D, L3, and R... 2 As defined in this article.

[0182] In other preferred embodiments, the suicide spacer is a portion having the following structure:

[0183] Lp is an enzymatically cleavable divalent peptide spacer, and D, L3, and R... 2 As defined herein. In some embodiments, D is a pharmaceutical portion containing a quaternized tertiary amine, wherein the ammonium cation is optionally present in a zwitterionic form or has a monovalent anionic counterion.

[0184] As used herein, the term "hydrophilic portion" refers to a portion having hydrophilic properties that increases the water solubility of the pharmaceutical portion (D) when it is attached to the linker group of the present invention. Examples of such hydrophilic groups include, but are not limited to, polyethylene glycol, polyalkylene glycol, sugars, oligosaccharides, polypeptides, and 1 to 3 [unclear text - possibly referring to specific compounds or components]. Group-substituted C2-C6 alkyl groups and polysarcosine, such as those having the formula Those, where n is an integer between 2 and 25; and R is H, -CH3 or -CH2CH2C(=O)OH.

[0185] In some embodiments, the hydrophilic portion comprises polyethylene glycol having the following formula: Where R is H, -CH3, -CH2CH2NHC(=O)OR a -CH2CH2NHC(=O)R a 、or -CH2CH2C(=O)OR a R' is OH, -OCH3, -CH2CH2NHC(=O)OR a -CH2CH2NHC(=O)R a , or -OCH2CH2C(=O)OR a , where R a It is H or optionally OH or C 1-4 alkoxy-substituted C 1-4 Alkyl group, and m and n are each integers between 2 and 25 (e.g., between 3 and 25). In some embodiments, the hydrophilic portion comprises...

[0186] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, or chimeric antibodies. These antibodies can have any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0187] The terms "antibody fragment," "antigen-binding fragment," or "functional fragment" refer to at least a portion of an antibody that retains the ability to specifically interact with an antigenic epitope (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (VL or VH), Camelidae VHH domains, multispecific antibodies formed from antibody fragments (such as bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, nanobodies, intracellular antibodies, bisomal antibodies, tripois antibodies, tetrasomal antibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted into peptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin peptide microantibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, for example, by a synthetic linker (e.g., a short flexible peptide linker) and are capable of being expressed as a single-chain peptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise stated, as used herein, the scFv may have VL and VH variable regions in any order relative to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0188] As used herein, the term “complementarity-determining region” or “CDR” refers to the sequence of amino acids within the variable region of an antibody that confers antigen specificity and binding affinity. For example, generally, there are three CDRs (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described in the following literature: Kabat et al., (1991), “Sequences of Proteins of Immunological Interest”, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB (Journal of Microbiology and Biotechnology) 273, 927-948 (“Chothia” numbering scheme), or combinations thereof, and Immunogenetics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). In combined Cabart and Josiah numbering schemes for a given CDR region (e.g., HC CDR1, HC CDR2, HC CDR3, LCCDR1, LC CDR2, or LC CDR3), in some embodiments, these CDRs correspond to amino acid residues defined as part of a Cabart CDR and amino acid residues defined as part of a Josiah CDR. As used herein, CDRs defined according to the “Josiah” numbering scheme are sometimes also referred to as “hypervariant rings”.

[0189] For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) (e.g., one or more insertions after position 35), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1) (e.g., one or more insertions after position 27), 50-56 (LCDR2), and 89-97 (LCDR3). As another example, according to Josiah, the CDR amino acids in VH are numbered 26-32 (HCDR1) (e.g., one or more insertions after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1) (e.g., one or more insertions after position 30), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Josiah, a CDR comprises, for example, amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL, or consists of them. According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (according to Kabat numbering). Under IMGT, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0190] The term "epitope" refers to any protein determinant capable of specifically binding to immunoglobulins or otherwise interacting with molecules. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acid or carbohydrate or sugar side chains, and may possess specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be "linear" or "conformal." The difference between conformational and linear epitopes lies in the fact that binding to a conformational epitope (rather than a linear epitope) is lost in the presence of denaturing solvents.

[0191] As used herein, the phrase "monoclonal antibody" or "monoclonal antibody composition" refers to polypeptides (including antibodies, bispecific antibodies, etc.) having substantially the same amino acid sequence or originating from the same genetic source. This term also includes formulations of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0192] As used herein, the phrase “human antibody” includes antibodies with variable regions in which both the frame region and the CDR region are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, for example, germline human sequences, or mutant forms of germline human sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, as described, for example, in Knappik et al. (2000. J Mol Biol [Journal of Molecular Biology] 296, 57-86). The structure and location of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Josiah numbering scheme, or a combination of Kabat and Josiah, and the ImMunoGenTics (IMGT) numbering scheme (see, for example, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), ed. Kabat et al.; Al Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest. Interest, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et al., (1997) J. Mal. Biol. 273:927-948; and Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol. 27, 55-77 (2003).

[0193] Human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., by introducing mutations to promote stability or production through random or site-specific mutagenesis in vitro, or through somatic mutations or conserved substitutions in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been transplanted into a human frame sequence.

[0194] As used herein, the phrase "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as antibodies isolated from animals (e.g., mice) (where the human immunoglobulin gene is transgenic or transchromosomally modified) or hybridomas prepared therefrom; antibodies isolated from host cells transformed to express human antibodies (e.g., from transfected tumors); antibodies isolated from a library of recombinant human antibodies; and antibodies prepared, expressed, generated, or isolated by any other means involving the splicing of all or part of the human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable regions, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be mutagenized in vitro (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences derived from and associated with human germline VH and VL sequences, but these sequences may not be naturally present in a human antibody germline library in vivo.

[0195] As used herein, the term "Fc region" refers to a polypeptide comprising at least a portion of the CH3, CH2, and hinge regions of the constant structural domains of an antibody. Optionally, the Fc region may include the CH4 domain present in some antibody classes. The Fc region may comprise the entire hinge region of the antibody's constant structural domain. In one embodiment, the invention comprises the Fc region and CH1 region of an antibody. In one embodiment, the invention comprises the Fc region and CH3 region of an antibody. In another embodiment, the invention comprises the Fc region, CH1 region, and Cκ / λ region from the antibody's constant structural domain. In one embodiment, the binding molecule of the invention comprises a constant region, for example, a heavy chain constant region. In one embodiment, such a constant region is modified compared to a wild-type constant region. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant structural domains (CH1, CH2, or CH3) and / or to one or more of the light chain constant region (CL) domains. Examples of modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.

[0196] As used herein, the term "binding specificity" refers to the ability of a single antibody binding site to react with one antigenic determinant and not with different antigenic determinants. The binding site of an antibody is located in the Fab region of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Antibody binding affinity is the strength of the reaction between a single antigenic determinant and a single binding site on the antibody. It is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antibody binding site.

[0197] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites via weak non-covalent forces; the more interactions, the stronger the affinity.

[0198] The term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within an antibody can be substituted with other amino acid residues from the same side chain family, and the altered antibody can be tested using the functional assays described herein.

[0199] The term "homologous" or "identical" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules (like two DNA molecules or two RNA molecules) or two polypeptide molecules. Subunit positions in both molecules are occupied by the same monomeric subunits; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at said position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., five positions in a polymer of ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous. The percentage of "sequence identity" can be determined by comparing two best-aligned sequences on a comparison window, where a fragment of amino acid sequence in the comparison window may contain additions or deletions (e.g., vacancies or overhangs) compared to a reference sequence (which does not contain additions or deletions) to achieve best alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions where the same amino acid residues appear in both sequences to generate the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. The output is the percentage of identity between the subject sequence and the query sequence. Taking into account the number of gaps and the length of each gap, the percentage of identity between two sequences is a function of the number of common positions shared by these sequences; these gaps need to be included for optimal alignment of the two sequences.

[0200] Sequence comparison and determination of identity percentage between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the Needleman and Wunsch ((1970) J. Mol. Biol. [Journal of Molecular Biology] 48:444-453) algorithm (which is incorporated into the GAP program in the GCG software package (available from www.gcg.com)) is used, employing a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the identity percentage between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available from www.gcg.com) is used, employing an NWSgapdna.CMP matrix with vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6 to determine the identity percentage between two nucleotide sequences. A particularly preferred set of parameters (and the parameters that should be used unless otherwise specified) is the Blossum 62 scoring matrix, in which the vacancy penalty is 12, the vacancy extension penalty is 4, and the shifted vacancy penalty is 5.

[0201] The percentage of identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS [Computer Applications in Biological Sciences] 4:11-17) (which has been incorporated into the ALIGN program (version 2.0)). The algorithm uses a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.

[0202] The nucleic acid and protein sequences described herein can be used as “query sequences” to search public databases for, for example, to identify other family members or related sequences. These searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of this invention. BLAST protein searches can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of this invention. For obtaining vacancy alignments for comparative purposes, vacancy BLAST (GappedBLAST) can be used as described in Altschul et al., (1997) Nucleic Acids Res. [Nucleic Acid Research] 25:3389-3402. When using the BLAST and empty BLAST procedures, the default parameters for the corresponding procedures (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0203] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of the compounds of the present invention with at least one and optionally more than one other pharmaceutically acceptable chemical component (such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, and / or excipient).

[0204] As used herein, the terms "optical isomer" or "stereoisomer" refer to any of the various stereoisomeric conformations that a given compound of the invention may exist in, including geometric isomers. It should be understood that substituents may be attached to the chiral center of a carbon atom. The term "chiral" refers to a molecule that has a non-overlapping property on its mirror-image partner, while the term "chiral" refers to a molecule that is overlapping on its mirror-image partner. Therefore, the invention includes enantiomers, diastereomers, or racemates of the said compounds. An "enantiomer" is a pair of stereoisomers that are non-overlapping mirror images of each other. A 1:1 mixture of enantiomer pairs is a "racemic" mixture. The term is used to denote racemic mixtures where appropriate. A "diastereomer" is a stereoisomer having at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry is defined according to the Cahn-Lngold-Prelog RS system. When the compound is a pure enantiomer, the stereochemistry at each chiral carbon can be described by R or S. The resolution of compounds with unknown absolute conformation can be specified as (+) or (-) depending on the direction (right-handed or left-handed) in which they rotate plane-polarized light with wavelengths of the sodium D line. Some of the compounds described herein contain one or more asymmetric centers or axes and thereby can produce enantiomers, diastereomers, and other stereoisomers that can be defined according to absolute stereochemistry as (R)- or (S)-.

[0205] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Consider its use in therapeutic or pharmaceutical compositions, except where any conventional carrier is incompatible with the active ingredient.

[0206] As used herein, the term "pharmaceutically acceptable salt" means a salt that does not eliminate the biological activity and properties of the compounds of the present invention and does not cause significant irritation to the subject to which it is administered.

[0207] As used herein, the term "subject" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs; rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. Typically, the subject is a human.

[0208] The term "subject in need of this treatment" refers to a subject who will benefit from this treatment biologically, medically, or in terms of quality of life.

[0209] As used herein, the term "treatment" for any disease or disorder, in one embodiment, means to alleviate the disease or disorder (i.e., to slow down or halt or reduce the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" means to relieve or reduce at least one bodily parameter, including those that cannot be identified by the patient. In yet another embodiment, "treatment" means to regulate the disease or disorder in a physical aspect (e.g., stabilization of identifiable symptoms), a physiological aspect (e.g., stabilization of bodily parameters), or both.

[0210] As used herein, the term “prevent, preventing, or prevention” for any disease or disorder refers to preventive treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0211] The terms "therapeutic effective dose" or "therapeutic effective amount" are interchangeable in meaning to refer to an amount sufficient to achieve the desired outcome (i.e., reducing or inhibiting enzyme or protein activity, alleviating symptoms, relieving symptoms or condition, delaying disease progression, reducing tumor size, inhibiting tumor growth, preventing metastasis, inhibiting or preventing viral, bacterial, fungal, or parasitic infections). In some embodiments, the therapeutic effective dose does not induce or cause undesirable side effects. In some embodiments, the therapeutic effective dose induces or causes side effects, but only those acceptable to a healthcare provider for the patient's condition. The therapeutic effective dose can be determined by initially administering a low dose and then incrementally increasing the dose until the desired effect is achieved. The "preventive effective dose" or "preventive effective amount" of the present invention molecules can prevent the onset of disease symptoms, including cancer-related symptoms. The "therapeutic effective dose" or "therapeutic effective amount" of the present invention molecules can lead to a reduction in the severity of disease symptoms, including cancer-related symptoms.

[0212] The compound names provided in this article were obtained using ChemBioDraw Ultra (version 14.0).

[0213] As used herein, the terms “a”, “the”, and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0214] Any formulas given herein are also intended to represent both unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures described by the formulas given herein, except that one or more atoms are replaced by atoms having a selected atomic weight or mass number. Isotopes that may be incorporated into the compounds of this invention include, for example, isotopes of hydrogen.

[0215] Connector-drug group

[0216] The linker-drug group of the present invention is a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0217]

[0218] in:

[0219] R 1 It is a reactive group;

[0220] L1 is a bridging spacer;

[0221] Lp is a divalent peptide spacer;

[0222] G-L2-A is a suicide spacer;

[0223] R 2 It is the hydrophilic part;

[0224] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0225] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0226] L3 is the spacer sub-part;

[0227] and

[0228] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0229] Certain aspects and examples of the connector-pharmaceutical group of the present invention are provided in the following list of enumerated embodiments. It should be understood that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0230] Example 1. A compound having formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0231] R 1 It is a reactive group;

[0232] L1 is a bridging spacer;

[0233] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0234] G-L2-A is a suicide spacer;

[0235] R 2 It is the hydrophilic part;

[0236] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0237] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0238] L3 is the spacer sub-part;

[0239] and

[0240] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0241] Example 2. A compound having formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0242] R 1 It is a reactive group;

[0243] L1 is a bridging spacer;

[0244] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0245] The The radical group is selected from:

[0246] in The * indicates the attachment point of the N or O component of the drug. The *** instruction and the attachment point of Lp;

[0247] R 2 It is the hydrophilic part;

[0248] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0249] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0250] L3 is the spacer sub-part;

[0251] and

[0252] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0253] Example 3. A compound having formula (I) and having the structure of formula (II):

[0254]

[0255] Or its pharmaceutically acceptable salt.

[0256] in:

[0257] R 1 It is a reactive group;

[0258] L1 is a bridging spacer;

[0259] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0260] R 2 It is the hydrophilic part;

[0261] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0262] L3 is the spacer sub-part;

[0263] and

[0264] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0265] Example 4. A compound having formula (I) as described in any one of Examples 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:

[0266] R 1 yes -ONH2, -NH2, N3, -SH、-SR 3 -SSR 4 , -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NHS(=O)2(CH=CH2), -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(O)NHNH2、

[0267]

[0268] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0269] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0270] *-C(=O)O(CH2) m SSC(R 3)2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;

[0271] *-C(=O)O(CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) m -**;

[0272] *-C(=O)(CH2) m NH(CH2) n C(=O)-**;*-C(=O)(CH2) m X1(CH2) m -**;

[0273] *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;

[0274] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0275] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0276] *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;

[0277] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(CH2) n -**;

[0278] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2)m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 Attachment point;

[0279] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0280] Each R 3 Independently selected from H and C1-C6 alkyl groups;

[0281] R 4 It is 2-pyridinyl or 4-pyridinyl;

[0282] Each R 5 Independently selected from H, C1-C6 alkyl groups, F, Cl, and -OH;

[0283] Each R 6 Independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2 and –OH;

[0284] Each R 7 Independently selected from H and C 1-6 Alkyl, fluorine, benzyloxy group substituted with -C(=O)OH, benzyl group substituted with -C(=O)OH, C group substituted with -C(=O)OH 1-4 Alkoxy groups and C groups substituted with -C(=O)OH 1-4 alkyl;

[0285] X1 is

[0286] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0287] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0288] Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0289] Lp is a divalent peptide spacer containing amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0290] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0291] L3 is a structure The spacer sub-part,

[0292] in

[0293] (i)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-**, -NH-, or -CH2N(R b )C(=O)CH2-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0294] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 The attachment point; or

[0295] (ii)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0296] X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene compounds -OC(O)NHS(O)2NH-*, ***-(CH2CH2O) n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*、or ***-CH2-triazolyl-C 1-4 Alkylene -OC(O)NHS(O)2NH-(CH2CH2O) n -*, where each n is independently 1, 2, or 3, the *** indicator of X is associated with the attachment point of W and the * indicator of X is associated with R. 2Attachment point;

[0297] and

[0298] The * indication of L3 and R 2 Attachment point;

[0299] and

[0300] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0301] Example 5. A compound having formula (I) as described in any one of Examples 1 to 4, or a pharmaceutically acceptable salt thereof, wherein:

[0302] R 1 yes -ONH2、

[0303] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 Attachment point;

[0304] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0305] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0306] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0307] Lp is selected from The divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G;

[0308] L3 is a structure The spacer sub-part,

[0309] in

[0310] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0311] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0312] and

[0313] The * indication of L3 and R 2 Attachment point;

[0314] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0315] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R aIndependently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0316] and

[0317] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0318] Example 6. A compound having formula (I) as described in any one of Examples 1 to 5, or a pharmaceutically acceptable salt thereof, wherein:

[0319] R 1 yes

[0320] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 Attachment point;

[0321] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0322] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0323] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0324] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0325] L3 is a structure The spacer sub-part,

[0326] in

[0327] W is -CH2O-**, -CH2N(R) b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0328] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0329] and

[0330] The * indication of L3 and R 2 Attachment point;

[0331] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0332] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0333] and

[0334] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0335] Example 7. A compound having formula (I) as described in any one of Examples 1 to 6, or a pharmaceutically acceptable salt thereof, wherein:

[0336] R 1 yes

[0337] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * indicator of L1 is associated with the attachment point of Lp and the ** indicator of L1 is associated with R 1 Attachment point;

[0338] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0339] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0340] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0341] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0342] L3 is a structure The spacer sub-part,

[0343] in

[0344] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0345] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0346] and

[0347] The * indication of L3 and R 2 Attachment point;

[0348] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0349] A is a bond or -OC (=O)*, where * indicates an attachment point to D;

[0350] and

[0351] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0352] Example 8. A compound having formula (I) as described in any one of Examples 1 to 7, or a pharmaceutically acceptable salt thereof, wherein:

[0353] R 1 yes

[0354] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t(CH2) n -, wherein the * indicator of L1 is associated with the attachment point of Lp and the ** indicator of L1 is associated with R 1 Attachment point;

[0355] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0356] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0357] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0358] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0359] L3 is a structure The spacer sub-part,

[0360] in

[0361] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0362] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0363] and

[0364] The * indication of L3 and R 2 Attachment point;

[0365] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0366] A is a bond or -OC (=O)*, where * indicates an attachment point to D;

[0367] and

[0368] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0369] Example 9. A compound having formula (I) as described in any one of Examples 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R 1 These are reactive groups selected from Table 1 or Table 2.

[0370] Example 10. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein:

[0371] R 1 yes -ONH2, -NH2, -N3、 -SH、-SR 3 -SSR 4 , -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NHS(=O)2(CH=CH2), -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(O)NHNH2、

[0372] Example 11. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein:

[0373] R 1 yes -ONH2, -NH2, -N3、 -SH、-SR 3 -SSR 4 , -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NHS(=O)2(CH=CH2), -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(O)NHNH2、

[0374] Example 12. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein:

[0375] R1 yes -ONH2、

[0376] Example 13. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein:

[0377] R 1 yes -ONH2、

[0378] Example 14. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R1 is

[0379] Example 15. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R 1 It is -ONH2.

[0380] Example 16. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein: R 1 yes

[0381] Example 17. A compound having formula (I) as described in any one of Examples 1 to 9, or a pharmaceutically acceptable salt thereof, wherein:

[0382] R 1 yes

[0383] Example 18. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0384] Or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0385] Example 19. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0386] Or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0387] Example 20. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0388] Or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0389] Example 21. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0390] Or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0391] Example 22. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0392] Or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0393] Example 23. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0394] Or a pharmaceutically acceptable salt thereof, wherein Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

[0395] Example 24. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0396] Or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0397] Example 25. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0398] Or a pharmaceutically acceptable salt thereof, wherein Xb is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

[0399] Example 26. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0400] Or its pharmaceutically acceptable salt.

[0401] Example 27. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0402] Or its pharmaceutically acceptable salt.

[0403] Example 28. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0404] Or its pharmaceutically acceptable salt.

[0405] Example 29. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0406] Or its pharmaceutically acceptable salt.

[0407] Example 30. A compound having formula (I) as described in any one of Examples 1 to 8, having the following structure:

[0408] Or its pharmaceutically acceptable salt.

[0409] Example 31. A compound having formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Examples 1 to 8, having the structure of a compound in any one of the tables 4A-4C included herein.

[0410] Example 32. A connector-drug group having formula (I), said connector having a structure of formula (V),

[0411]

[0412] in

[0413] L1 is a bridging spacer;

[0414] Lp is a divalent peptide spacer;

[0415] G-L2-A is a suicide spacer;

[0416] R 2 It is the hydrophilic part;

[0417] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0418] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each Ra Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the ** of A indicates the attachment point with L2.

[0419] and

[0420] L3 is the spacer sub-part.

[0421] Example 33. The connector as described in Example 32, wherein:

[0422] L1 is a bridging spacer;

[0423] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0424] G-L2-A is a suicide spacer;

[0425] R 2 It is the hydrophilic part;

[0426] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0427] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of A is the attachment point with L2.

[0428] and

[0429] L3 is the spacer sub-part.

[0430] Example 34. The connector as described in Example 32 or 33, wherein:

[0431] L1 is a bridging spacer;

[0432] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0433] The The radical group is selected from:

[0434] in The * indicates the attachment point of the N or O component of the drug. The *** instruction and the attachment point of Lp;

[0435] R 2 It is the hydrophilic part;

[0436] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0437] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the ** of A indicates the attachment point with L2.

[0438] and

[0439] L3 is the spacer sub-part.

[0440] Example 35. The connector as described in any one of Examples 32 to 34, wherein:

[0441] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0442] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0443] *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;

[0444] *-C(=O)O(CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) m -**;

[0445] *-C(=O)(CH2) mNH(CH2) n C(=O)-**;*-C(=O)(CH2) m X1(CH2) m -**;

[0446] *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;

[0447] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0448] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0449] *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;

[0450] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(CH2) n -**;

[0451] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicates the attachment point of L1 with Lp;

[0452] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0453] Each R 3Independently selected from H and C1-C6 alkyl groups;

[0454] X1 is

[0455] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0456] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0457] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0458] Lp is a divalent peptide spacer containing amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0459] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of A is the attachment point of L2;

[0460] L3 is a structure The spacer sub-part,

[0461] in

[0462] (i)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b-**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-**, -NH-, or -CH2N(R b )C(=O)CH2-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0463] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 The attachment point; or

[0464] (ii)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0465] X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、

[0466] ***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*

[0467] ***-(CH2CH2O) n -C(O)NHS(O)2NH-*、

[0468] ***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*、or ***-CH2-triazolyl-C 1-4 Alkylene -OC(O)NHS(O)2NH-(CH2CH2O) n -*, where each n is independently 1, 2, or 3, the *** indicator of X is associated with the attachment point of W and the * indicator of X is associated with R. 2 Attachment point;

[0469] and

[0470] The * indication of L3 and R 2 Attachment point.

[0471] Example 36. The connector as described in any one of Examples 32 to 35, wherein:

[0472] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0473] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0474] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0475] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0476] Lp is selected from The divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G;

[0477] L3 is a structure The spacer sub-part,

[0478] in

[0479] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0480] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0481] and

[0482] The * indication of L3 and R 2 Attachment point;

[0483] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0484] and

[0485] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of A is the attachment point of L2.

[0486] Example 37. The connector as described in any one of Examples 32 to 36, wherein:

[0487] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0488] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0489] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0490] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0491] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0492] L3 is a structure The spacer sub-part,

[0493] in

[0494] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0495] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0496] and

[0497] The * indication of L3 and R 2 Attachment point;

[0498] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0499] and

[0500] A is a bond, **-OC (=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of A is the attachment point of L2.

[0501] Example 38. The connector as described in any one of Examples 32 to 37, wherein:

[0502] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0503] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0504] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0505] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0506] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0507] L3 is a structure The spacer sub-part,

[0508] in

[0509] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 -**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b-**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0510] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0511] and

[0512] The * indication of L3 and R 2 Attachment point;

[0513] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0514] and

[0515] A is a key or **-OC (=O)-, where the ** indicates the attachment point of L2.

[0516] Example 39. The connector as described in any one of Examples 32 to 38, wherein:

[0517] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0518] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0519] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0520] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0521] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0522] L3 is a structure The spacer sub-part,

[0523] in

[0524] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0525] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0526] and

[0527] The * indication of L3 and R 2 Attachment point;

[0528] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. The hydrophilic moiety of a C2-C6 alkyl group substituted with a functional group;

[0529] and

[0530] A is a key or **-OC (=O)-, where the ** indicates the attachment point of L2.

[0531] Example 40. A connector having type (V) and having the structure of type (VI),

[0532]

[0533] in

[0534] L1 is a bridging spacer;

[0535] Lp is a divalent peptide spacer;

[0536] R 2 It is the hydrophilic part;

[0537] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl, or C3-C8 cycloalkyl.

[0538] and

[0539] L3 is the spacer sub-part.

[0540] Example 41. The connector as described in Example 40, wherein:

[0541] L1 is a bridging spacer;

[0542] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0543] R 2 It is the hydrophilic part;

[0544] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl, or C3-C8 cycloalkyl.

[0545] and

[0546] L3 is the spacer sub-part.

[0547] Example 42. The connector as described in Example 40 or 41, wherein:

[0548] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0549] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0550] *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;

[0551] *-C(=O)O(CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) m -**;

[0552] *-C(=O)(CH2) m NH(CH2) n C(=O)-**;*-C(=O)(CH2) m X1(CH2) m -**;

[0553] *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;

[0554] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0555] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0556] *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;

[0557] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(CH2) n -**;

[0558] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicates the attachment point of L1 with Lp;

[0559] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, or the hydrophilic portion of polysarcosine;

[0560] Each R 3 Independently selected from H and C1-C6 alkyl groups;

[0561] X1 is

[0562] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0563] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0564] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0565] Lp is a divalent peptide spacer containing amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0566] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a Independently selected from H, C1-C6 alkyl, or C3-C8 cycloalkyl;

[0567] L3 is a structure The spacer sub-part,

[0568] in

[0569] (i)W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-**, -NH-, or -CH2N(R b )C(=O)CH2-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0570] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 The attachment point; or

[0571] (ii)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR bC(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0572] X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene compounds -OC(O)NHS(O)2NH-*, ***-(CH2CH2O) n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*、or ***-CH2-triazolyl-C 1-4 Alkylene -OC(O)NHS(O)2NH-(CH2CH2O) n -*, where each n is independently 1, 2, or 3, the *** indicator of X is associated with the attachment point of W and the * indicator of X is associated with R. 2 Attachment point;

[0573] and

[0574] The * indication of L3 and R 2 Attachment point.

[0575] Example 43. The connector as described in any one of Examples 40 to 42, wherein:

[0576] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0577] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0578] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0579] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0580] Lp is selected from A divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group;

[0581] L3 is a structure The spacer sub-part,

[0582] in

[0583] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0584] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0585] and

[0586] The * indication of L3 and R 2 Attachment point;

[0587] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, or the hydrophilic portion of polysarcosine;

[0588] and

[0589] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a It is independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl.

[0590] Example 44. The connector as described in any one of Examples 40 to 43, wherein:

[0591] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp;

[0592] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0593] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0594] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0595] Lp is selected from (ValCit) divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group;

[0596] L3 is a structure The spacer sub-part,

[0597] in

[0598] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0599] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0600] and

[0601] The * indication of L3 and R 2 Attachment point;

[0602] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. The hydrophilic portion of C2-C6 alkyl groups or polysarcosine groups that are substituted with functional groups;

[0603] and

[0604] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a)2C(R a )2N(CH3)C(=O)-, where each R a It is independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl.

[0605] Example 45. The connector as described in any one of Examples 40 to 44, wherein:

[0606] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - wherein the * indicator of L1 is the attachment point of Lp;

[0607] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0608] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0609] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0610] Lp is selected from (ValCit) divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group;

[0611] L3 is a structure The spacer sub-part,

[0612] in

[0613] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 -**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NRb C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0614] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0615] and

[0616] The * indication of L3 and R 2 Attachment point;

[0617] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, or the hydrophilic portion of polysarcosine;

[0618] and

[0619] A is a bond or -OC (=O)-.

[0620] Example 46. The connector as described in any one of Examples 40 to 45, wherein:

[0621] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp;

[0622] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0623] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0624] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0625] Lp is selected from (ValCit) divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group;

[0626] L3 is a structure The spacer sub-part,

[0627] in

[0628] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0629] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0630] and

[0631] The * indication of L3 and R 2 Attachment point;

[0632] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, or the hydrophilic portion of polysarcosine;

[0633] and

[0634] A is a bond or -OC (=O)-.

[0635] Example 47. The connector as described in any one of Examples 32 to 46, having the following structure:

[0636] in

[0637] R is H, -CH3, or -CH2CH2C(=O)OH.

[0638] Example 48. The connector as described in any one of Examples 32 to 46, having the following structure:

[0639] in

[0640] R is H, -CH3, or -CH2CH2C(=O)OH.

[0641] Example 49. The connector as described in any one of Examples 32 to 46, having the following structure:

[0642] in

[0643] R is H, -CH3, or -CH2CH2C(=O)OH.

[0644] Example 50. The connector as described in any one of Examples 32 to 46, having the following structure:

[0645] in

[0646] Each R is independently selected from H, -CH3, or -CH2CH2C(=O)OH.

[0647] Example 51. The connector as described in any one of Examples 32 to 46, having the following structure:

[0648] in

[0649] Each R is independently selected from H, -CH3, or -CH2CH2C(=O)OH.

[0650] Example 52. The connector as described in any one of Examples 32 to 46, having the following structure:

[0651] in

[0652] Xa is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

[0653] Example 53. The connector as described in any one of Examples 32 to 46, having the following structure:

[0654] in

[0655] R is H, -CH3, or -CH2CH2C(=O)OH.

[0656] Example 54. The connector as described in any one of Examples 32 to 46, having the following structure:

[0657] in

[0658] Xb is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

[0659] Example 55. The connector as described in any one of Examples 32 to 46, having the following structure:

[0660]

[0661] Example 56. The connector as described in any one of Examples 32 to 46, having the following structure:

[0662]

[0663] Example 57. The connector as described in any one of Examples 32 to 46, having a structure

[0664]

[0665] Example 58. The connector as described in any one of Examples 32 to 46, having the following structure:

[0666]

[0667] Example 59. The connector as described in any one of Examples 32 to 46, having the following structure:

[0668]

[0669] For illustrative purposes, the general reaction schemes described herein provide possible pathways for synthesizing the compounds of the present invention and key intermediates. For a more detailed description of each reaction step, see the Examples section below. Although specific starting materials and reagents are described in the schemes and discussed below, other starting materials and reagents can be readily substituted to provide a variety of derivatives and / or reaction conditions. Furthermore, many compounds prepared by the methods described below can be further modified using conventional chemistry well known to those skilled in the art, according to this disclosure.

[0670] For example, the general synthesis of compounds having formula (II) is shown in Scheme 1 below.

[0671] Option 1

[0672]

[0673] The antibody-drug conjugate of the present invention

[0674] The present invention provides antibody-drug conjugates, also referred to herein as immunoconjugates, which comprise a linker containing one or more hydrophilic moieties.

[0675] The antibody-drug conjugate of the present invention has the structure of formula (III):

[0676]

[0677] in:

[0678] Ab is an antibody or a fragment thereof;

[0679] R 100 It is a coupling group;

[0680] L1 is a bridging spacer;

[0681] Lp is a divalent peptide spacer;

[0682] G-L2-A is a suicide spacer;

[0683] R 2 It is the hydrophilic part;

[0684] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0685] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0686] L3 is the spacer sub-part;

[0687] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0688] and

[0689] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0690] Certain aspects and examples of the antibody-drug conjugates of the present invention are provided in the following list of enumerated embodiments. It should be understood that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0691] Example 60. An immunoconjugate having formula (III), wherein:

[0692] Ab is an antibody or a fragment thereof;

[0693] R 100 It is a coupling group;

[0694] L1 is a bridging spacer;

[0695] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0696] G-L2-A is a suicide spacer;

[0697] R 2 It is the hydrophilic part;

[0698] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0699] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0700] L3 is the spacer sub-part;

[0701] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0702] and

[0703] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0704] Example 61. An immunoconjugate having formula (III) as described in Example 60, wherein:

[0705] Ab is an antibody or a fragment thereof;

[0706] R 100 It is a coupling group;

[0707] L1 is a bridging spacer;

[0708] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0709] The The radical group is selected from:

[0710] in The * indicates the attachment point of the N or O component of the drug. The *** instruction and the attachment point of Lp;

[0711] R 2 It is the hydrophilic part;

[0712] L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene;

[0713] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0714] L3 is the spacer sub-part;

[0715] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0716] and

[0717] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0718] Example 62. An immunoconjugate having formula (III) as described in any one of Examples 60 to 61, having the structure of formula (IV),

[0719]

[0720] in:

[0721] Ab is an antibody or a fragment thereof;

[0722] R 100 It is a coupling group;

[0723] L1 is a bridging spacer;

[0724] Lp is a divalent peptide spacer containing one to four amino acid residues;

[0725] R 2 It is the hydrophilic part;

[0726] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0727] L3 is the spacer sub-part;

[0728] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0729] and

[0730] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0731] Example 63. An immunoconjugate having formula (III) as described in any one of Examples 60 to 62, wherein:

[0732] Ab is an antibody or a fragment thereof;

[0733] R 100 yes -S-, -C(=O)-, -ON=***, -NHC(=O)CH2-***, -S(=O)2CH2CH2-***, -(CH2)2S(=O)2CH2CH2-***, -NHS(=O)2CH2CH 2- ***, -NHC(=O)CH2CH2-***, -CH2NHCH2CH2-***, -NHCH2CH2-***, Wherein R 100 The *** instruction and the attachment point of Ab;

[0734] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0735] *-C(=O)(CH2) m-**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0736] *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;

[0737] *-C(=O)O(CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) m -**;

[0738] *-C(=O)(CH2) m NH(CH2) n C(=O)-**;*-C(=O)(CH2) m X1(CH2) m -**;

[0739] *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;

[0740] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0741] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0742] *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;

[0743] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2)n X1(CH2) n -**;

[0744] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 100 Attachment point;

[0745] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, and the hydrophilic portion of polysarcosine;

[0746] Each R 3 Independently selected from H and C1-C6 alkyl groups;

[0747] R 4 It is 2-pyridinyl or 4-pyridinyl;

[0748] Each R 5 Independently selected from H, C1-C6 alkyl groups, F, Cl, and -OH;

[0749] Each R 6 Independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2 and –OH;

[0750] Each R 7 Independently selected from H and C 1-6 Alkyl, fluorine, benzyloxy group substituted with -C(=O)OH, benzyl group substituted with -C(=O)OH, C group substituted with -C(=O)OH 1-4 Alkoxy groups and C groups substituted with -C(=O)OH 1-4 alkyl;

[0751] X1 is

[0752] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0753] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0754] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0755] Lp is a divalent peptide spacer containing amino acid residues selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0756] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0757] L3 is a structure The spacer sub-part,

[0758] in

[0759] (i)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b-**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-**, -NH-, or -CH2N(R b )C(=O)CH2-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0760] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 The attachment point; or

[0761] (ii)W is -CH2O-**, -CH2N(R) b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0762] X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene compounds -OC(O)NHS(O)2NH-*, ***-(CH2CH2O)n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*、or ***-CH2-triazolyl-C 1-4 Alkylene -OC(O)NHS(O)2NH-(CH2CH2O) n -*, where each n is independently 1, 2, or 3, the *** indicator of X is associated with the attachment point of W and the * indicator of X is associated with R. 2 Attachment point;

[0763] and

[0764] The * indication of L3 and R 2 Attachment point;

[0765] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0766] and

[0767] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0768] Example 64. An immunoconjugate having formula (III) as described in any one of Examples 60 to 63, wherein:

[0769] Ab is an antibody or a fragment thereof;

[0770] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab;

[0771] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 100 Attachment point;

[0772] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0773] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0774] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0775] Lp is selected from The divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G;

[0776] L3 is a structure The spacer sub-part,

[0777] in

[0778] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0779] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0780] and

[0781] The * indication of L3 and R 2 Attachment point;

[0782] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, and the hydrophilic portion of polysarcosine;

[0783] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0784] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0785] and

[0786] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0787] Example 65. An immunoconjugate having formula (III) as described in any one of Examples 60 to 64, wherein:

[0788] Ab is an antibody or a fragment thereof;

[0789] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab;

[0790] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n - where the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 100 Attachment point;

[0791] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0792] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0793] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0794] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0795] L3 is a structure The spacer sub-part,

[0796] in

[0797] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0798] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0799] and

[0800] The * indication of L3 and R 2 Attachment point;

[0801] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, or composed of 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, and the hydrophilic portion of polysarcosine;

[0802] A is a key, -OC (=O)-*, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-*or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-*, where each R a Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, and the * of A indicates the attachment point of D;

[0803] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0804] and

[0805] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0806] Example 66. An immunoconjugate having formula (III) as described in any one of Examples 60 to 65, wherein:

[0807] Ab is an antibody or a fragment thereof;

[0808] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab;

[0809] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * indicator of L1 is associated with the attachment point of Lp and the ** indicator of L1 is associated with R 100Attachment point;

[0810] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0811] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0812] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0813] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0814] L3 is a structure The spacer sub-part,

[0815] in

[0816] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 -**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0817] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0818] and

[0819] The * indication of L3 and R 2 Attachment point;

[0820] R2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, and the hydrophilic portion of polysarcosine;

[0821] A is a bond or -OC (=O)*, where * indicates an attachment point to D;

[0822] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0823] and

[0824] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Example 67. An immunoconjugate having formula (III) as described in any one of Examples 60 to 66, wherein:

[0825] Ab is an antibody or a fragment thereof;

[0826] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab;

[0827] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * indicator of L1 is associated with the attachment point of Lp and the ** indicator of L1 is associated with R 100 Attachment point;

[0828] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0829] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0830] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

[0831] Lp is selected from (ValCit) is a divalent peptide spacer, wherein the * of Lp indicates the attachment site with L1 and the ** of Lp indicates the attachment site with the -NH- group of G.

[0832] L3 is a structure The spacer sub-part,

[0833] in

[0834] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0835] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0836] and

[0837] The * indication of L3 and R 2 Attachment point;

[0838] R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups, and the hydrophilic portion of polysarcosine;

[0839] A is a bond or -OC (=O)*, where * indicates an attachment point to D;

[0840] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[0841] and

[0842] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0843] Example 68. An immunoconjugate having formula (III) as described in any one of Examples 60 to 62, wherein R 100 It is a coupling group.

[0844] Example 69. An immunoconjugate having formula (III) as described in any one of Examples 60 to 63, wherein

[0845] R 100 yes -S-, -C(=O)-, -ON=***, -NHC(=O)CH2-***, -S(=O)2CH2CH2-***, -(CH2)2S(=O)2CH2CH2-***, -NHS(=O)2CH2CH 2- ***, -NHC(=O)CH2CH2-***, -CH2NHCH2CH2-***, -NHCH2CH2-***, Where R 100 The *** instruction and the attachment point of Ab.

[0846] Example 70. An immunoconjugate having formula (III) as described in any one of Examples 60 to 63, wherein

[0847] R 100 yes -S-, -C(=O)-, -ON=***, -NHC(=O)CH2-***, -S(=O)2CH2CH2-***, -(CH2)2S(=O)2CH2CH2-***, -NHS(=O)2CH2CH 2- ***, -NHC(=O)CH2CH2-***, -CH2NHCH2CH2-***, -NHCH2CH2-***, Where R 100 The *** instruction and the attachment point of Ab.

[0848] Example 71. An immunoconjugate having formula (III) as described in any one of Examples 60 to 63, wherein

[0849] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab.

[0850] Example 72. An immunoconjugate having formula (III) as described in any one of Examples 60 to 63, wherein

[0851] R 100 yes Wherein R 100 The *** instruction and the attachment point of Ab.

[0852] Example 73. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0853] in

[0854] R is H, -CH3, or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0855] Example 74. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0856] in

[0857] R is H, -CH3, or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0858] Example 75. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0859] in

[0860] R is H, -CH3, or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0861] Example 76. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0862] in

[0863] Each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0864] Example 77. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0865] in

[0866] Each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0867] Example 78. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0868] Where Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0869] Example 79. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0870] Where R is H, -CH3 or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0871] Example 80. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0872]

[0873] in

[0874] Xb is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0875] Example 81. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0876] Where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0877] Example 82. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure: Where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0878] Example 83. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0879] Where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0880] Example 84. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0881] Where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0882] Example 85. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72, having the following structure:

[0883] Where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0884] Certain aspects and examples of the linker-drug groups, linkers, and antibody-drug conjugates of the present invention are provided in the following list of additional enumerated embodiments. It should be understood that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0885] Example 86. A compound having formula (I) as described in any one of Examples 1 to 2, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 39; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 61, wherein:

[0886] G is Wherein the * of G indicates the attachment point of L2, the ** of G indicates the attachment point of L3, and the *** of G indicates the attachment point of Lp.

[0887] Example 87. A compound having formula (I) as described in any one of Examples 1 to 2, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 39; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 61, wherein:

[0888] G is Wherein the * of G indicates the attachment point of L2, the ** of G indicates the attachment point of L3, and the *** of G indicates the attachment point of Lp.

[0889] Example 88. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein:

[0890] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0891] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0892] *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;

[0893] *-C(=O)O(CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) m -**;

[0894] *-C(=O)(CH2) m NH(CH2) n C(=O)-**;*-C(=O)(CH2) m X1(CH2) m -**;

[0895] *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;

[0896] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0897] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0898] *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;

[0899] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(CH2) n -**;

[0900] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0901] Example 89. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein:

[0902] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;

[0903] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0904] *-C(=O)(CH2) m NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) n C(=O)-**;

[0905] *-C(=O)(CH2) m NHC(=O)(CH2) n -**;

[0906] *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;

[0907] *-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0908] Example 90. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein:

[0909] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2)n -**;

[0910] *-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;

[0911] *-C(=O)(CH2) m NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) n C(=O)-**; or

[0912] *-C(=O)(CH2) m NHC(=O)(CH2) n -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0913] Example 91. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein:

[0914] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -** or *-C(=O)NH((CH2) m O) t (CH2) n -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0915] Example 92. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein L1 is *-C(=O)(CH2). m O(CH2) m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0916] Example 93. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein L1 is *-C(=O)((CH2)). m O) t (CH2) n -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0917] Example 94. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein L1 is *-C(=O)(CH2). m -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R. 1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0918] Example 95. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72, wherein L1 is *-C(=O)NH((CH2) m O) t (CH2) n -**, wherein the * indicator of L1 is the attachment point of Lp, and the ** indicator of L1 is the attachment point of R.1 (If present) the attachment point or the **indicator of L1 and R 100 (If it exists) an attachment point.

[0919] Example 96. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein Lp is a divalent peptide spacer, for example, an enzymatically cleavable spacer.

[0920] Example 97. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein Lp is a divalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0921] Example 98. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein Lp is a divalent peptide spacer comprising one to four amino acid residues (e.g., two to four amino acid residues).

[0922] Example 99. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein Lp is a divalent peptide spacer comprising one to four amino acid residues, each of which is independently selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0923] Example 100. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0924] Lp is selected from The divalent peptide spacer, wherein the * of Lp indicates the attachment site of L1 and the ** of Lp indicates the attachment site of the -NH- group of formula (II) or the ** of Lp indicates the attachment site of G of formula (I).

[0925] Example 101. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0926] Lp is The * of Lp indicates the attachment point of L1 and the ** of Lp indicates the attachment point of the -NH- group of formula (II) or the ** of Lp indicates the attachment point of G of formula (I).

[0927] Example 102. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0928] Lp is The * of Lp indicates the attachment point of L1 and the ** of Lp indicates the attachment point of the -NH- group of formula (II) or the ** of Lp indicates the attachment point of G of formula (I).

[0929] Example 103. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0930] Lp is (ValAla), wherein the * of Lp indicates the attachment point of L1 and the ** of Lp indicates the attachment point of the -NH- group of formula (II) or the ** of Lp indicates the attachment point of G of formula (I).

[0931] Example 104. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0932] Lp is The * of Lp indicates the attachment point of L1 and the ** of Lp indicates the attachment point of the -NH- group of formula (II) or the ** of Lp indicates the attachment point of G of formula (I).

[0933] Example 105. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 95, wherein:

[0934] Lp is The * of Lp indicates the attachment point of L1 and the ** of Lp indicates the attachment point of the -NH- group of formula (II) or the ** of Lp indicates the attachment point of G of formula (I).

[0935] Example 106. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 105, wherein L2 is a bond, methylene, neopentyl or C2-C3 alkenyl.

[0936] Example 107. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 105, wherein L2 is a bond or a methylene group.

[0937] Example 108. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 105, wherein L2 is a bond.

[0938] Example 109. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 105, wherein L2 is a methylene group.

[0939] Example 110. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein:

[0940] A is a bond, -OC (=O)-, -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or-OC(=O)N(CH3)C(R a )2C(R a )2N(CH3)C(=O)-, where each R a It is independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl.

[0941] Example 111. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein A is a bond or -OC (=O).

[0942] Example 112. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein A is a bond.

[0943] Example 113. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein A is -OC (=O).

[0944] Example 114. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein:

[0945] A is

[0946] Example 115. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 109, wherein:

[0947] A is -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)- or -OC(=O)N(CH3)C(R) a )2C(R a )2N(CH3)C(=O)-, where each R a It is independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl.

[0948] Example 116. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, a linker having formula (V) as described in any one of Examples 32 to 46, and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein L3 is a spacer portion.

[0949] Example 117. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0950] L3 is a structure The spacer sub-part,

[0951] in

[0952] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、NHC(=O)C(R b)2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0953] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0954] and

[0955] The * indication of L3 and R 2 Attachment point.

[0956] Example 118. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0957] L3 is a structure The spacer sub-part,

[0958] in

[0959] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -NHC(=O)CH2NH-**, NHC(=O)CH2NHC(=O)-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2)-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0960] X is a key;

[0961] and

[0962] The * indication of L3 and R 2 Attachment point.

[0963] Example 119. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0964] L3 is a structure The spacer sub-part,

[0965] in

[0966] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b-**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0967] X is a triazole group, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0968] and

[0969] The * indication of L3 and R 2 Attachment point.

[0970] Example 120. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0971] L3 is a structure The spacer sub-part,

[0972] in

[0973] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-C(=O)NR b -**、-C(=O)NH-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or -NH-, where each R bIndependently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0974] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0975] and

[0976] The * indication of L3 and R 2 Attachment point.

[0977] Example 121. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0978] L3 is a structure The spacer sub-part,

[0979] in

[0980] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0981] X is a bond, a triazole group, or ***-CH2-triazole-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0982] and

[0983] The * indication of L3 and R 2 Attachment point.

[0984] Example 122. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0985] L3 is a structure The spacer sub-part,

[0986] in

[0987] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0988] X is a key;

[0989] and

[0990] The * indication of L3 and R 2 Attachment point.

[0991] Example 123. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0992] L3 is a structure The spacer sub-part,

[0993] in

[0994] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[0995] X is a triazole group, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[0996] and

[0997] The * indication of L3 and R 2 Attachment point.

[0998] Example 124. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 115, wherein:

[0999] L3 is a structure The spacer sub-part,

[1000] in

[1001] W is -CH2O-**, -CH2N(R) b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**, where each R b Independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl, wherein the **indication of W is the attachment point of X;

[1002] X is ***-CH2-triazolyl-*, wherein the *** of X indicates the attachment site of W and the * of X indicates the attachment site of R. 2 Attachment point;

[1003] and

[1004] The * indication of L3 and R 2 Attachment point.

[1005] Example 125. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is the hydrophilic part.

[1006] Example 126. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharides, polypeptides, and 1 to 3 [unclear]. Group-substituted C2-C6 alkyl groups and the hydrophilic portion of polysarcosine.

[1007] Example 127. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It's sugar.

[1008] Example 128. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is an oligosaccharide.

[1009] Example 129. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is a polypeptide.

[1010] Example 130. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is a polyalkylene glycol.

[1011] Example 131. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It has the structure -(O(CH2)) m ) t R' is a polyalkylene glycol, wherein R' is OH, OCH3 or OCH2CH2C(=O)OH, m is 1-10 and t is 4-40.

[1012] Example 132. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It has the structure -((CH2) m O) t R”- a polyalkylene glycol, wherein R” is H, CH3 or CH2CH2C(=O)OH, m is 1-10 and t is 4-40.

[1013] Example 133. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It is polyethylene glycol.

[1014] Example 134. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It has the structure -(OCH2CH2) t R' is polyethylene glycol, wherein R' is OH, OCH3 or OCH2CH2C(=O)OH and t is 4-40.

[1015] Example 135. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein R 2 It has the structure -(CH2CH2O) t R”-polyethylene glycol, wherein R” is H, CH3 or CH2CH2C(=O)OH and t is 4-40.

[1016] Example 136. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein:

[1017] R 2 yes (where n is an integer between 1 and 6) Wherein R 2 The * or wavy line indicates the attachment point with X or L3.

[1018] Example 137. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein:

[1019] R 2 yes Wherein R 2 The * indicates the attachment point of X or L3.

[1020] Example 138. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein:

[1021] R 2 yes Where R 2 The * indicates the attachment point of X or L3.

[1022] Example 139. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 124, wherein:

[1023] R 2 yes Where R 2 The * indicates the attachment point of X or L3.

[1024] Example 140. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 139, wherein each R 3Independently selected from H and C1-C6 alkyl groups.

[1025] Example 141. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 139, wherein each R 3 It is H.

[1026] Example 142. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 139, wherein each R 3 It is independently selected from C1-C6 alkyl groups.

[1027] Example 143. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 142, wherein:

[1028] X1 is

[1029] Example 144. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 142, wherein:

[1030] X1 is

[1031] Example 145. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 144, wherein:

[1032] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[1033] Example 146. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 144, wherein:

[1034] Each m is independently selected from 1, 2, 3, 4, and 5.

[1035] Example 147. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 144, wherein:

[1036] Each m is independently selected from 1, 2, and 3.

[1037] Example 148. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 147, wherein:

[1038] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[1039] Example 149. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 147, wherein:

[1040] Each n is independently selected from 1, 2, 3, 4, and 5.

[1041] Example 150. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 147, wherein:

[1042] Each n is independently selected from 1, 2, and 3.

[1043] Example 151. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 150, wherein:

[1044] Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.

[1045] Example 152. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 150, wherein:

[1046] Each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.

[1047] Example 153. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 150, wherein:

[1048] Each t is independently selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[1049] Example 154. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof; a linker having formula (V) as described in any one of Examples 32 to 46; and an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 150, wherein:

[1050] Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

[1051] Example 155. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[1052] Example 156. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[1053] Example 157. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[1054] Example 158. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[1055] Example 159. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7 or 8.

[1056] Example 160. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3, 4, 5 or 6.

[1057] Example 161. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1, 2, 3 or 4.

[1058] Example 162. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 1 or 2.

[1059] Example 163. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 2.

[1060] Example 164. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 4.

[1061] Example 165. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 6.

[1062] Example 166. An immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 154, wherein y is 8.

[1063] Example 167. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is the pharmaceutical part.

[1064] Example 168. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein

[1065] D is a pharmaceutical part containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical part.

[1066] Example 169. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion.

[1067] Example 170. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein

[1068] D is a hydrophobic pharmaceutical moiety containing N or O, wherein D is connected to A via a direct bond from A to the N or O of the pharmaceutical moiety (e.g., when connected to A, D may be a quaternary ammonium).

[1069] Example 171. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 7.

[1070] Example 172. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 6.

[1071] Example 173. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 5.

[1072] Example 174. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 4.

[1073] Example 175. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 3.

[1074] Example 176. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 1.5 to 2.

[1075] Example 177. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 2 to 7.

[1076] Example 178. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 2 to 6.

[1077] Example 179. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 2 to 5.

[1078] Example 180. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 2 to 4.

[1079] Example 181. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is a hydrophobic pharmaceutical portion having a SlogP value of 2 to 3.

[1080] Example 182. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is olistatin.

[1081] Example 183. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is

[1082] Example 184. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is not an MCL-1 inhibitor.

[1083] Example 185. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is not a BCL-2 inhibitor.

[1084] Example 186. A compound having formula (I) as described in any one of Examples 1 to 17 or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein D is not a BCL-XL inhibitor.

[1085] Example 187. A compound having formula (I) as described in any one of Examples 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate having formula (III) as described in any one of Examples 60 to 72 or any one of Examples 86 to 166, wherein the linker in formula (I) (i.e., the portion without D) or the linker in formula (III) (i.e., the portion connecting Ab and D) is selected from the linkers from L2 to L208 disclosed herein (e.g., in Tables 4A-4C), such as The wavy line indicates the attachment point with D.

[1086] Example 188. A connector having a connector-drug group having a structure of formula (V) as described in any of Examples 32 to 46, derived from any connector selected from L2 to L208 described herein, for example, such as (Source: L2) (Source: L71) (derived from L179), and (Derived from L208), where the wavy line indicates the attachment point with D and the * indicates the attachment point with the antibody or its fragment.

[1087] Joining methods

[1088] The present invention provides various methods for conjugating the linker-drug group of the present invention with an antibody or antibody fragment to produce an antibody-drug conjugate comprising a linker having one or more hydrophilic moieties.

[1089] The general reaction scheme for forming antibody-drug conjugates having formula (III) is shown in the following scheme 2:

[1090] Option 2

[1091]

[1092] Wherein: RG2 is a reactive group, which is compatible with R 1 Group reactions to form the corresponding R 100 Groups (such groups are shown in Table 1). D, R 1 L1, Lp, Ab, y, and R 100 As defined in this article.

[1093] Scheme 3 further illustrates this general method for forming antibody-drug conjugates having formula (III), wherein the antibody comprises R 1 The reactive group (RG2) reacts with the group (as defined herein) via R 100 A group (as defined herein) covalently attaches the linker-drug group to the antibody. For illustrative purposes only, Scheme 3 illustrates an antibody having four RG2 groups.

[1094] Option 3

[1095]

[1096] In one aspect, the linker-drug group is conjugated to the antibody via a modified cysteine ​​residue in the antibody (see, for example, WO2014 / 124316). Scheme 4 illustrates a method for forming an antibody-drug conjugate having formula (III), wherein a free thiol group generated from an engineered cysteine ​​residue in the antibody is conjugated to R. 1 Group (where R) 1 (for maleimide) reaction, via R 100 Group (where R) 100 The linker-drug group is covalently attached to the antibody (using a succinimide ring). For illustrative purposes only, Scheme 4 shows an antibody having four free thiol groups.

[1097] Option 4

[1098]

[1099] On the other hand, the linker-drug group is conjugated to the antibody via a lysine residue in the antibody. Scheme 5 illustrates a method for forming an antibody-drug conjugate having formula (III), wherein a free amino group from a lysine residue in the antibody is conjugated to R... 1 Group (where R) 1 (For NHS esters, pentafluorophenyl or tetrafluorophenyl) react to via R 100 Group (where R) 100 An amide is used to covalently attach the linker-drug group to the antibody. For illustrative purposes only, Scheme 5 shows an antibody having four amino groups.

[1100] Option 5

[1101]

[1102] On the other hand, the linker-drug group is conjugated to the antibody via the formation of an oxime bridge at the naturally occurring disulfide bridge of the antibody. An oxime bridge is formed by first reducing the interchain disulfide bridge of the antibody and then bridging it again using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). This is then reacted with a linker-drug group containing hydroxylamine to form an oxime bond (oxime bridge) that attaches the linker-drug group to the antibody (see, for example, WO2014 / 083505). Scheme 6 illustrates this method for forming an antibody-drug conjugate having formula (III).

[1103] Option 6

[1104]

[1105] A general reaction scheme for forming antibody-drug conjugates having formula (IV) is shown in the following scheme 7:

[1106] Option 7

[1107]

[1108] Wherein: RG2 is a reactive group, which is compatible with R 1 Group reactions to form the corresponding R 100 Groups (such groups are shown in Table 1). D, R 1 L1, Lp, Ab, y, and R 100 As defined in this article.

[1109] Scheme 8 further illustrates this general method for forming antibody-drug conjugates having formula (IV), wherein the antibody comprises R 1 The reactive group (RG2) reacts with the group (as defined herein) via R 100 A group (as defined herein) covalently attaches the linker-drug group to the antibody. For illustrative purposes only, Scheme 8 illustrates an antibody having four RG2 groups.

[1110] Option 8

[1111]

[1112] In one aspect, the linker-drug group is conjugated to the antibody via a modified cysteine ​​residue in the antibody (see, for example, WO2014 / 124316). Scheme 9 illustrates a method for forming an antibody-drug conjugate having formula (IV), wherein a free thiol group generated from an engineered cysteine ​​residue in the antibody is conjugated to R... 1 Group (where R) 1(for maleimide) reaction, via R 100 Group (where R) 100 The linker-drug group is covalently attached to the antibody (which is a succinimide ring). For illustrative purposes only, Scheme 9 shows an antibody having four free thiol groups.

[1113] Option 9

[1114]

[1115] On the other hand, the linker-drug group is conjugated to the antibody via a lysine residue in the antibody. Scheme 10 illustrates a method for forming an antibody-drug conjugate having formula (IV), wherein a free amino group from a lysine residue in the antibody is conjugated to R... 1 Group (where R) 1 (For NHS esters, pentafluorophenyl or tetrafluorophenyl) react to via R 100 Group (where R) 100 An amide is used to covalently attach the linker-drug group to the antibody. For illustrative purposes only, Scheme 10 shows an antibody having four amino groups.

[1116] Option 10

[1117]

[1118] On the other hand, the linker-drug group is conjugated to the antibody via the formation of an oxime bridge at the naturally occurring disulfide bridge of the antibody. An oxime bridge is formed by first reducing the interchain disulfide bridge of the antibody and then bridging it again using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). This is then reacted with a linker-drug group containing hydroxylamine to form an oxime bond (oxime bridge) that attaches the linker-drug group to the antibody (see, for example, WO2014 / 083505). Scheme 11 illustrates this method for forming an antibody-drug conjugate having formula (IV).

[1119] Option 11

[1120]

[1121] Schemes for evaluating certain aspects of the analytical methodologies for the antibody conjugates of the present invention are also provided. Such analytical methodologies and results can demonstrate that the conjugates possess advantageous properties, such as making them easier to manufacture, easier to administer to patients, more effective for patients, and / or potentially safer. One example is the determination of molecular size by size exclusion chromatography (SEC), where the amount of the desired antibody species in the sample is determined relative to the amount of high molecular weight contaminants (e.g., dimers, polymers, or aggregated antibodies) or low molecular weight contaminants (e.g., antibody fragments, degradation products, or individual antibody chains) present in the sample. Typically, higher amounts of monomers and lower amounts of, for example, aggregated antibodies are desired due to, for example, the effect of aggregates on other properties of the antibody sample, such as, but not limited to, clearance, immunogenicity, and toxicity. A further example is the determination of hydrophobicity by hydrophobic interaction chromatography (HIC), where the hydrophobicity of the sample is evaluated relative to a set of standard antibodies with known properties. Low hydrophobicity is generally desirable because hydrophobicity affects other properties of antibody samples, such as, but not limited to, aggregation, aggregation over time, adhesion to surfaces, hepatotoxicity, clearance, and pharmacokinetic exposure. See Damle, NK, Nat Biotechnol. [Nature Biotechnology] 2008; 26(8):884-885; Singh, SK, Pharm Res. [Pharmaceutical Research] 2015; 32(11):3541-71. When measured by hydrophobic interaction chromatography, a higher hydrophobicity index score (i.e., faster elution from the HIC column) reflects lower hydrophobicity of the conjugate. As shown in the examples below, most of the antibody conjugates tested showed a hydrophobicity index greater than 0.8. In some embodiments, antibody conjugates with a hydrophobicity index of 0.8 or greater, as determined by hydrophobic interaction chromatography, are provided.

[1122] Antibody

[1123] This invention provides antibody conjugates comprising antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to antigens (e.g., tumor antigens). The antibodies or antibody fragments (e.g., antigen-binding fragments) of this invention include, but are not limited to, human monoclonal antibodies or fragments thereof isolated as described in the examples.

[1124] In some embodiments, the present invention provides antibody conjugates comprising an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to P-cadherin, said antibody or antibody fragment (e.g., an antigen-binding fragment) comprising a VH domain having an amino acid sequence having SEQ ID NO: 7, 27, 47, 67, 87, 107, or 154. In some embodiments, the present invention also provides antibody conjugates comprising an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to P-cadherin, said antibody or antibody fragment (e.g., an antigen-binding fragment) comprising a VH CDR having an amino acid sequence having any of the VH CDRs listed in Table 3 below. In particular embodiments, the present invention provides antibody conjugates comprising an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to P-cadherin, said antibody comprising (or alternatively, consisting of) one, two, three, four, five, or more VH CDRs having an amino acid sequence having any of the VH CDRs listed in Table 3 below.

[1125] This invention provides antibody conjugates comprising antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to P-cadherin, said antibodies or antibody fragments (e.g., antigen-binding fragments) comprising a VL domain having an amino acid sequence having SEQ ID NO: 17, 37, 57, 77, 97, 117, or 166. This invention also provides antibody conjugates comprising antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to P-cadherin, said antibodies or antibody fragments (e.g., antigen-binding fragments) comprising a VL CDR having an amino acid sequence having any of the VL CDRs listed in Table 3 below. In particular, this invention provides antibody conjugates comprising antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to P-cadherin, said antibodies or antibody fragments (e.g., antigen-binding fragments) comprising (or alternatively, consisting of) one, two, three, or more VL CDRs having an amino acid sequence having any of the VL CDRs listed in Table 3 below.

[1126] Other antibodies or antibody fragments of the present invention (e.g., antigen-binding fragments) comprise amino acids that have been mutated but have at least 60%, 70%, 80%, 90%, or 95% identity in the CDR region with the CDR region depicted in the sequences described in Table 3. In some embodiments, the antibody comprises a mutated amino acid sequence wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR region have been mutated when compared to the CDR region depicted in the sequences described in Table 3.

[1127] This invention also provides antibody conjugates comprising an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising modifications in a constant region of the heavy chain, light chain, or both heavy and light chains, wherein specific amino acid residues have been mutated to cysteine, referred herein also as “CysMab” or “Cys” antibodies. As discussed above, a drug moiety can be conjugated to cysteine ​​residues on an antibody in a site-specific manner and with control over the amount of the drug moiety (“DAR-controlled”). For example, cysteine ​​modifications of antibodies for site-specific control of immune conjugation are disclosed in WO2014 / 124316, the entire text of which is incorporated herein by reference.

[1128] In some embodiments, the antibody has been modified at positions 152 and 375 on the heavy chain, where the positions are defined according to the EU numbering system. That is, the modifications are E152C and S375C. In other embodiments, the antibody has been modified at position 360 on the heavy chain and position 107 on the κ light chain, where the positions are defined according to the EU numbering system. That is, the modifications are K360C and K107C. For example, the positions of these mutations are illustrated in the context of the constant regions of the human IgG1 heavy chain and κ light chain in SEQ ID NO:148-150 of Table 3. Cysteine ​​modifications from wild-type sequences are shown underlined throughout Table 3.

[1129] This invention also provides nucleic acid sequences for the VH, VL, full-length heavy chain, and full-length light chain of antibodies that specifically bind to P-cadherin. These nucleic acid sequences can be optimized for expression in mammalian cells.

[1130] Table 3. Examples of the anti-P-cadherin antibody of the present invention

[1131]

[1132]

[1133]

[1134]

[1135]

[1136]

[1137]

[1138]

[1139]

[1140]

[1141]

[1142]

[1143]

[1144]

[1145]

[1146]

[1147]

[1148]

[1149]

[1150]

[1151]

[1152]

[1153]

[1154]

[1155]

[1156]

[1157]

[1158] Other antibodies of the present invention include those in which the amino acid or the nucleic acid encoding the amino acid has been mutated, but has at least 60%, 70%, 80%, 90%, or 95% identity with the sequences described in Table 3. In some embodiments, when compared with the variable regions depicted in the sequences described in Table 3, 1, 2, 3, 4, or 5 amino acids in the variable region have been mutated, but while retaining substantially the same therapeutic activity as the antibodies listed in Table 3.

[1159] In some embodiments, antibodies or antibody fragments (e.g., antigen-binding fragments) that can be used in the immunoconjugates of the present invention comprise modified or engineered antibodies, such as antibodies modified to introduce one or more cysteine ​​residues as sites for conjugation to a drug moiety (Junutula JR et al.: Nat Biotechnol 2008, 26: 925-932). In one embodiment, the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of one or more amino acids with cysteine ​​at the positions described herein. The sites for cysteine ​​substitution are in the constant region of the antibody and are therefore applicable to a variety of antibodies, and the sites are selected to provide stable and homogeneous conjugates. Modified antibodies or fragments may have two or more cysteine ​​substitutions, and these substitutions may be used in combination with other antibody modification and conjugation methods as described herein. Methods for inserting cysteine ​​residues at specific sites on antibodies are known in the art, see, for example, Lyons et al., (1990) Protein Eng., 3:703-708; WO 2011 / 005481; WO 2014 / 124316. In some embodiments, the modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine ​​at constant regions selected from the following positions on the heavy chain of the antibody or antibody fragment: positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, ​​390, 392, 398, 400, and 422, and wherein said positions are numbered according to the EU system. In some embodiments, the modified antibody or antibody fragment comprises substitutions of one or more amino acids with cysteine ​​at constant regions selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of the light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system, and wherein the light chain is a human κ light chain. In some embodiments, the modified antibody or antibody fragment thereof comprises a combination of substitutions of two or more amino acids with cysteine ​​at constant regions of the antibody, wherein the combination comprises substitutions at position 375, position 152, position 360, or position 107 of the antibody heavy chain, and wherein the positions are numbered according to the EU system.In some embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​in its constant region, wherein said substitution is at position 375, position 152, position 360, position 107, position 165, or position 159 of the antibody light chain, and said positions are numbered according to the EU system, and said light chain is a κ chain. In a particular embodiment, the modified antibody or antibody fragment thereof comprises a combination of substitutions of two amino acids with cysteine ​​in its constant region, wherein the combination comprises substitutions at positions 375 and 152 of the antibody heavy chain, said positions are numbered according to the EU system. In a particular embodiment, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​at position 360 of the antibody heavy chain, said position is numbered according to the EU system. In other particular embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​at position 107 of the antibody light chain, and said position is numbered according to the EU system, and said light chain is a κ chain. Exemplary embodiments of these positions are shown in the constant region sequences disclosed in SEQ ID NO:148, 149, and 150. Specific embodiments of these positions are disclosed with respect to anti-P-cadherin antibody sequences in SEQ ID NO:130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147.

[1160] Because each of these antibodies can bind to P-cadherin, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding said amino acid sequences) can be "mixed and matched" to produce other P-cadherin-binding antibodies of the present invention. Such "mixed and matched" P-cadherin-binding antibodies can be tested using binding assays known in the art (e.g., ELISA, and other assays described in the Examples section). When these chains are mixed and matched, the VH sequence from a particular VH / VL pair should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Likewise, the VL sequence from a particular VH / VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence. Therefore, in one aspect, the present invention provides an antibody conjugate comprising an isolated monoclonal antibody or an antigen-binding region thereof, said monoclonal antibody or the antigen-binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 27, 47, 67, 87, and 107; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 37, 57, 77, 97, and 117; wherein said antibody specifically binds to P-cadherin.

[1161] In another aspect, the present invention provides antibody conjugates comprising (i) an isolated monoclonal antibody having: a full-length heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 29, 49, 69, 89, and 109, optimized for expression in cells of a mammalian expression system; and a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 39, 59, 79, 99, and 119, optimized for expression in mammalian cells; or (ii) a functional protein comprising its antigen-binding portion.

[1162] In another aspect, the present invention provides antibody conjugates comprising P-cadherin-binding antibodies, said P-cadherin-binding antibodies comprising heavy and light chains CDR1, CDR2, and CDR3, or combinations thereof, as described in Table 3. The amino acid sequences of the VH CDR1 of said antibody are shown in SEQ ID NO: 1, 21, 41, 61, 81, and 101. The amino acid sequences of the VH CDR2 of said antibody are shown in SEQ ID NO: 2, 22, 42, 62, 82, and 102. The amino acid sequences of the VH CDR3 of said antibody are shown in SEQ ID NO: 3, 23, 43, 63, 83, and 103. The amino acid sequences of the VL CDR1 of said antibody are shown in SEQ ID NO: 11, 31, 51, 71, 91, and 111. The amino acid sequences of the VL CDR2 of said antibody are shown in SEQ ID NO: 12, 32, 52, 72, 92, and 112. The amino acid sequence of the VL CDR3 of the antibody is shown in SEQ ID NO: 13, 33, 53, 73, 93, and 113.

[1163] Given that each of these antibodies can bind to P-cadherin and that antigen-binding specificity is primarily provided by CDR1, 2, and 3 regions, VH CDR1, CDR2, and CDR3 sequences and VL CDR1, CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs from different antibodies can be mixed and matched). Such “mixed and matched” P-cadherin-binding antibodies can be tested using binding assays known in the art and those described in the examples (e.g., ELISA). When mixing and matching VH CDR sequences, the CDR1, CDR2, and / or CDR3 sequences from a specific VH sequence should be replaced with one or more structurally similar CDR sequences. Similarly, when mixing and matching VL CDR sequences, the CDR1, CDR2, and / or CDR3 sequences from a specific VL sequence should be replaced with one or more structurally similar CDR sequences. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be generated by replacing one or more VH and / or VL CDR region sequences with a structurally similar sequence from the CDR sequence shown herein for use with the monoclonal antibody of the present invention.

[1164] Therefore, the present invention provides isolated monoclonal antibodies or their antigen-binding regions thereof, said isolated monoclonal antibodies or their antigen-binding regions comprising: heavy chain CDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 21, 41, 61, 81, and 101; heavy chain CDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 22, 42, 62, 82, and 102; heavy chain CDR3, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 23, 43, 63, 83, and 103; light chain CDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 31, 51, 71, 91, and 111; light chain CDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 32, 52, 72, 92, and 112; and light chain CDR3, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 21, 41, 61, 81, and 103 ...1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 21, 41, 61, 81, and 103; and light chain CDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 21, 41, 61, 81, and 103; and light chain CDR3, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 21, 41, 61, 81, and 103 The amino acid sequence of the group consisting of NO: 13, 33, 53, 73, 93, and 113; wherein the antibody specifically binds to P-cadherin.

[1165] In certain embodiments, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO:1; the heavy chain CDR2 of SEQ ID NO:2; the heavy chain CDR3 of SEQ ID NO:3; the light chain CDR1 of SEQ ID NO:11; the light chain CDR2 of SEQ ID NO:12; and the light chain CDR3 of SEQ ID NO:13.

[1166] In another specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO:21; the heavy chain CDR2 of SEQ ID NO:22; the heavy chain CDR3 of SEQ ID NO:23; the light chain CDR1 of SEQ ID NO:31; the light chain CDR2 of SEQ ID NO:32; and the light chain CDR3 of SEQ ID NO:33.

[1167] In yet another embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO:41; the heavy chain CDR2 of SEQ ID NO:42; the heavy chain CDR3 of SEQ ID NO:43; the light chain CDR1 of SEQ ID NO:51; the light chain CDR2 of SEQ ID NO:52; and the light chain CDR3 of SEQ ID NO:53.

[1168] In another embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO:61; the heavy chain CDR2 of SEQ ID NO:62; the heavy chain CDR3 of SEQ ID NO:63; the light chain CDR1 of SEQ ID NO:71; the light chain CDR2 of SEQ ID NO:72; and the light chain CDR3 of SEQ ID NO:73.

[1169] In another specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO:81; the heavy chain CDR2 of SEQ ID NO:82; the heavy chain CDR3 of SEQ ID NO:83; the light chain CDR1 of SEQ ID NO:91; the light chain CDR2 of SEQ ID NO:92; and the light chain CDR3 of SEQ ID NO:93.

[1170] In another specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises the heavy chain CDR1 of SEQ ID NO: 101; the heavy chain CDR2 of SEQ ID NO: 102; the heavy chain CDR3 of SEQ ID NO: 103; the light chain CDR1 of SEQ ID NO: 111; the light chain CDR2 of SEQ ID NO: 112; and the light chain CDR3 of SEQ ID NO: 113.

[1171] In some embodiments, the antibody that specifically binds to P-cadherin is the antibody or antibody fragment (e.g., antigen-binding fragment) described in Table 3.

[1172] 2. Further changes to the Fc region framework

[1173] The immunoconjugates of the present invention may comprise a modified antibody or an antigen-binding fragment thereof, said modified antibody or antigen-binding fragment thereof further comprising modifications to framework residues within the VH and / or VL, for example, to improve the properties of the antibody. In some embodiments, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to “revert” one or more framework residues to their corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence of the derived antibody. To restore the framework region sequence to its germline conformation, the somatic mutation can be “reverted” to a germline sequence, for example, by site-directed mutagenesis. Such “reverted” antibodies are also intended to be covered by the present invention.

[1174] Another type of framework modification involves mutating one or more residues within the framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 to Carr et al.

[1175] In addition to modifications performed within the frame or CDR region, or as alternatives to modifications performed within the frame or CDR region, the antibodies of the present invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, thereby again altering one or more functional properties of the antibody. Each of these embodiments is described in more detail below.

[1176] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Changing the number of cysteine ​​residues in the CH1 hinge region can, for example, promote the assembly of light and heavy chains or increase or decrease antibody stability.

[1177] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, resulting in the antibody having impaired binding to staphylococcal protein A (SpA) relative to the native Fc-hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[1178] In other embodiments, the effector function of the antibody is altered by substituting at least one amino acid residue into the Fc region with different amino acid residues. For example, one or more amino acids can be substituted with different amino acid residues to give the antibody altered affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand that alters affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in U.S. Patents 5,624,821 and 5,648,260 to Winter et al.

[1179] In another embodiment, one or more amino acids selected from amino acid residues may be substituted with different amino acid residues, such that the antibody exhibits altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[1180] In another embodiment, one or more amino acid residues are altered to change the antibody's ability to fix complement. This method is described, for example, in PCT disclosure WO 94 / 29351 by Bodmer et al. In specific embodiments, one or more amino acids of the antibody or its antigen-binding fragment of the present invention are replaced by one or more variant amino acid residues. Variant amino acid residues also include, but are not limited to, constant regions of the heavy chains of IgG1, IgG2, and IgG3 subclasses and constant regions of the light chains of the κ isotype, as described by Jefferis et al., MAbs. [Monoclonal Antibodies] 1:332-338 (2009).

[1181] In yet another embodiment, the Fc region is modified to increase the antibody-mediated antibody-dependent cytotoxicity (ADCC) and / or the antibody affinity for the Fcγ receptor is increased by modifying one or more amino acids. This method is described, for example, in Presta's PCT publication WO 00 / 42072. Furthermore, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[1182] In yet another embodiment, the glycosylation of the antibody is modified. For example, glycosylated-free antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for an "antigen". Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Such glycosylation can increase the antibody's affinity for the antigen. This method is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[1183] Alternatively or concurrently, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosylated residues or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. This type of glycosylation modification can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a dysfunctional FUT8 gene encoding fucosylation, such that antibodies expressed in this cell line exhibit hypofucosylation. Presta describes a variant CHO cell line, Lecl3, in PCT Publication WO 03 / 035835, which exhibits reduced ability to attach fucose to Asn(297)-linked carbohydrates and also results in hypofucosylation of antibodies expressed in said host cells (see also Shields et al., (2002), J. Biol. Chem. 277:26733-26740). Umana et al. describe a cell line engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminetransferase III (GnTIII)) in PCT Publication WO 99 / 54342, which results in antibodies expressed in the engineered cell line exhibiting increased bipartite GlcNac structures, leading to increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[1184] In another embodiment, the antibody is modified to increase its biological half-life. Various methods can be employed. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F as described by Ward in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be modified within the CH1 or CL region to contain a salvage receptor-binding epitope with two loops of the CH2 domain derived from the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[1185] 3. Antibody production

[1186] Antibodies and their antibody fragments (e.g., antigen-binding fragments) can be generated by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, through hybridoma or recombinant generation. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[1187] This disclosure further provides polynucleotides encoding the antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity-determining regions as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 8, 28, 48, 68, 88, 108, and 151. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 18, 38, 58, 78, 98, 118, and 153.

[1188] In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide of SEQ ID NO: 10, 30, 50, 70, 90, 110, or 152. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide of SEQ ID NO: 20, 40, 60, 80, 100, 120, or 154.

[1189] The polynucleotides of this invention can encode only the variable region sequence of an antibody. They can also encode both the variable and constant regions of an antibody. Some polynucleotide sequences encode polypeptides comprising the variable regions of the heavy and light chains of an exemplary anti-P-cadherin antibody. Some other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of the heavy and light chains of an antibody, respectively.

[1190] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding antibodies or their binding fragments (e.g., sequences described in the examples below). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. [Enzymatic Methods] 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. [Enzymatic Methods] 68:109, 1979; the diethylphosphite method of Beaucage et al., Tetra. Lett. [Tetrahedral Letters] 22:1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations into polynucleotide sequences via PCR can be performed as described in the following literature, for example: PCR Technology: Principles and Applications for DNA Amplification, HAErlich (ed.), Freeman Press, New York, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[1191] This invention also provides expression vectors and host cells for generating the antibodies described herein. A variety of expression vectors can be used to express polynucleotides encoding antibody chains or binding fragments. Both virus-based and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, augmentation vectors (typically having expression cassettes for expressing proteins or RNA), and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Nature Genetics] 15:345, 1997). For example, non-viral vectors that can be used to express anti-P-cadherin polynucleotides and peptides in mammalian (e.g., human) cells include pThioHis A, B, and C, and pcDNA. TM3.1 His, pEBVHisA, B, and C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include those based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40, papillomaviruses, HBP EBV, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., ibid.; Smith, Annu. Rev. Microbiol. [Annual Review of Microbiology] 49:807, 1995; and Rosenfeld et al., Cell [Cell] 68:143, 1992.

[1192] The choice of expression vector depends on the intended host cell for expression. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operatively linked to a polynucleotide encoding an anti-P-cadherin antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of the inserted sequence under conditions other than induction. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Culture of transformed organisms can be scaled up under non-inducible conditions without favoring a population of host cells that better tolerate the coding sequence of their expression product. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the antibody chain or fragment. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequences. Furthermore, expression efficiency can be improved by including enhancers suitable for the cell system in use (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol. 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[1193] Expression vectors can also provide a secretion signal sequence location to form a fusion protein with a polypeptide encoded by an inserted antibody sequence. More commonly, the inserted antibody sequence is linked to the signal sequence prior to inclusion in the vector. Vectors used to receive sequences encoding variable domains of the light and heavy chains of anti-P-cadherin antibodies sometimes also encode constant regions or portions thereof. Such vectors allow the expression of the variable region as a fusion protein with the constant region, resulting in the production of the complete antibody or fragment thereof. Typically, such constant regions are human.

[1194] The host cell used to carry and express the antibody chain can be prokaryotic or eukaryotic. *Escherichia coli* is a prokaryotic host suitable for cloning and expressing the polynucleotides of the present invention. Other suitable microbial hosts include bacilli (such as *Bacillus subtilis*) and other Enterobacteriaceae (such as *Salmonella* and *Serratia*) and various *Pseudomonas* species. Expression vectors can also be prepared in these prokaryotic hosts, typically containing expression control sequences (e.g., origin of replication) compatible with the host cell. Furthermore, any number of well-known promoters will be available, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or promoter systems derived from bacteriophage λ. Promoters typically optionally use operon sequences to control expression and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express the antibody peptides of the present invention. Insect cells combined with baculovirus vectors can also be used.

[1195] In some preferred embodiments, mammalian host cells are used to express and produce the antibody peptides of the present invention. For example, they may be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones as described in the examples) or mammalian cell lines carrying exogenous expression vectors (e.g., SP2 / 0 myeloma cells exemplified below). These include any normal, morbid, or normal or aberrant immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cells for peptide expression is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, New York, NY, 1987. Expression vectors for mammalian host cells may contain expression control sequences, such as origin of replication, promoters, and enhancers (see, for example, Queen et al., Immunol. Rev. [Immunology Review] 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or modulotropic. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as the human CMV immediate early promoter), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[1196] The methods used to introduce expression vectors containing the target polynucleotide sequence vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (see Sambrook et al., 2012, *Molecular Cloning: A Laboratory Manual*, Volumes 1–4, Cold Spring Harbor Press, New York). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, impingement, virions, immunoliposomes, polycationic:nucleic acid conjugates, naked DNA, artificial viral particles, fusions with the herpesvirus structural protein VP22 (Elliot and O'Hare, *Cell* 88:223, 1997), drug-enhanced DNA uptake, and in vitro transduction. For long-term, high-yield production of recombinant proteins, stable expression is generally desired. For example, the expression vector of this invention, containing a viral origin of replication or an endogenous expression element and a selective marker gene, can be used to prepare cell lines that stably express antibody chains or binding fragments. After introducing the vector, cells can be grown in enrichment medium for 1-2 days, after which they are transferred to selective medium. The purpose of the selective marker is to confer selective resistance, and its presence allows cells that successfully express the introduced sequence to grow in selective medium. Tissue culture techniques suitable for the cell type can be used to proliferate resistant, stably transfected cells.

[1197] Therapeutic uses and treatment methods

[1198] The provided antibody conjugates can be used in a variety of applications, including but not limited to cancer treatment. In some embodiments, the antibody conjugates provided herein can be used to inhibit tumor growth, reduce tumor volume, induce differentiation, and / or reduce tumorigenicity. The methods of use can be in vitro, ex vivo, or in vivo.

[1199] In some embodiments, this document provides methods for treating, preventing, or alleviating a disease (e.g., cancer) in a subject (e.g., a human patient) by administering any of the antibody conjugates described herein to a subject. Use of the antibody conjugates of the present invention for treating or preventing a disease in a subject (e.g., a human patient) is also provided. Use of the antibody conjugates in treating or preventing a disease in a subject is further provided. In some embodiments, antibody conjugates for preparing a medicament for treating or preventing a disease in a subject are provided. In some embodiments, the disease treated with the antibody conjugate is cancer.

[1200] In one respect, the immunoconjugates described herein can be used to treat solid tumors. Examples of solid tumors include malignancies of various organ systems, such as sarcomas, adenocarcinomas, germ cell tumors, and carcinomas, such as those affecting the liver, lungs, breast, lymph nodes, biliary tract (e.g., colon), genitourinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, small cell lung cancers, non-small cell lung cancers, small bowel cancers, and esophageal cancers. In one embodiment, the cancer is melanoma, such as advanced melanoma. Examples of other cancers that can be treated include: bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, anal cancer, peritoneal cancer, and stomach cancer (or gastric cancer). Cancer, esophageal cancer, salivary gland cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, malignant glioma, neuroblastoma, cervical cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), children Solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, epidermoid carcinomas, squamous cell carcinomas, T-cell lymphomas, environmentally induced cancers (including asbestos-induced cancers), and combinations of the aforementioned cancers.

[1201] On the other hand, the immunoconjugates described herein can be used to treat hematologic cancers. Hematologic cancers include leukemia, lymphoma, and malignant lymphoproliferative disorders affecting the blood, bone marrow, and lymphatic system.

[1202] Leukemia can be classified into acute leukemia and chronic leukemia. Acute leukemia can be further classified into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemia"), a diverse set of hematological disorders characterized by ineffective production (or developmental abnormalities) of bone marrow blood cells and a combined risk of transformation into AML.

[1203] Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

[1204] In some embodiments, the cancer is a hematologic cancer, including but not limited to, for example, acute leukemia, including but not limited to, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), and acute lymphoblastic leukemia (“ALL”); one or more chronic leukemias, including but not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL); other hematologic cancers or hematologic disorders, including but not limited to, B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell leukemia, etc. This includes various hematologic disorders such as cellular tumors, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndromes and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenström macroglobulinemia, and "preleukemia," a collection of hematologic symptoms resulting from ineffective production (or developmental abnormalities) of bone marrow hematopoietic cells. Other diseases associated with tumor antigen expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative disorders expressing tumor antigens as described herein. The methods and compositions of this invention can also be used to treat or prevent metastatic lesions of the aforementioned cancers.

[1205] In some embodiments, the cancer is characterized by cells expressing a target tumor antigen, and the antibody of the antibody conjugate, or an antibody fragment (e.g., an antigen-binding fragment), binds to the target tumor antigen. In some embodiments, the immunoconjugate as described herein may comprise an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a tumor antigen (e.g., a tumor antigen as described herein). Methods for detecting the presence or overexpression of such tumor antigens are known to those skilled in the art and include methods such as immunohistocompatibility (IHC) assays using antibodies that specifically bind to tumor antigens, and methods for detecting the RNA expression level of tumor antigens.

[1206] In some embodiments, the tumor antigen is selected from one or more of the following targets: receptor tyrosine-protein kinase ERBB2 (Her2 / neu); receptor tyrosine-protein kinase ERBB3 (Her3); receptor tyrosine-protein kinase ERBB4 (Her4); epidermal growth factor receptor (EGFR); E-cadherin; P-cadherin; cadherin 6; cathepsin D; estrogen receptor; progesterone receptor; CA125; CA15-3; CA19-9; P-glycoprotein (CD243); CD2; CD19; CD20; CD22; CD24; CD27; CD30; CD37; CD38; CD40; CD44v6; CD45; CD47; CD52; CD56; CD70; CD71; CD79a; CD79b; CD72; CD97; CD179a; CD123; CD137; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); Epidermal growth factor receptor variant III (EGFRvIII); Ganglioside G2 (GD2); Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); blood Endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); folate receptor α; neural cell adhesion molecule (NCAM); prostate enzymes; prostatic acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); liver ligand B2; fibroblast activation protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAIX); proteasome (Macropain) subunit, β-type, 9 (LMP2); glycoprotein 100 (gp100);Oncogene fusion protein (bcr-abl) composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl); tyrosinase; liver ligand type A receptor 2 (EphA2); fucose GM1; sialic acid Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 associated (TEM7R); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C5 group, member D (GPRC5D); chromosome X reading frame 61 (CXO) RF61); Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide moiety of globoH glycosylceramide (GloboH); Breast differentiation antigen (NY-BR-1); Adrenaline receptor β3 (ADRB3); Pantothecin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Olfactory receptor 51E2 (OR51E2); TCRγ substitution reading frame protein (TARP); Nephroblastoma protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; Tumor protein p53 (p53); p53 mutant; Prostate-specific protein; Surviving protein; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or galactosin 8), T-cell 1-recognized melanoma antigen (MelanA or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTE) RT); sarcoma translocation breakpoint; melanoma cell apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-associated protein 2 (TRP-2);Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like protein (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); pairing box protein Pax-5 (PAX5); pro-acromial protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase ankylosing protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal insufficiency protein 1 (RU1); renal insufficiency protein 2 (RU2); Legumin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecular-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); Mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan-3 (GPC3); and Immunoglobulin λ-like polypeptide 1 (IGLL1); CD184; LGR5; AXL; RON; CD35 2 / SLAMf6; KAAG-1; 5T4; c-Met; ITGA3; Endothelial sialic acid protein; CD166; SAIL (c15orf54); NaPi2b; DLL3; CD133; FZD7; Antiadhesin; PD-L1; SLITRK6; Ligin-4; FGFR2; FGFR3; FGFR4; CEACAM1; CEACAM5; CD74; STEAP-1; PMEL17; Muc16; FcRH5; TENB2; Ly6E; ETBR; 158P1D7; 161P2F10B; 191p4d12; 162p1e6; Notch3; PTK7; and EFNA4.

[1207] Antigens that support tumors

[1208] In some embodiments, the immunoconjugates described herein may comprise an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein).

[1209] In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells, antigen-presenting cells, or myeloid-derived suppressor cells (MDSCs). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can suppress T cell proliferation and activation. In some embodiments, the stromal cell antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activating protein (FAP), and tendinin. In embodiments, the MDSC antigen is selected from one or more of the following: CD33, CD11b, C14, CD15, and CD66b. Therefore, in some embodiments, the tumor-supporting antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activating protein (FAP) or tendinin, CD33, CD11b, C14, CD15, and CD66b.

[1210] The antibody conjugates described in this paper are also envisioned for the treatment of a variety of non-malignant diseases or disorders, such as inflammatory bowel disease (IBD), gastrointestinal ulcers, Menetrier's disease, hepatitis B, hepatitis C, secretory adenoma or protein loss syndrome, kidney disorders, angiogenesis disorders, eye diseases (such as age-related macular degeneration, presumed ocular histoplasmosis syndrome, or age-related macular degeneration), bone-related disorders (such as osteoarthritis, rickets, and osteoporosis), systemic hyperviscosity syndrome, Osler-Weber-Rendudisease, chronic obstructive pulmonary disease, or edema following burns, trauma, radiation, stroke, hypoxia, or local ischemia, diabetic nephropathy, and Paget's disease. Diseases, photoaging (e.g., caused by ultraviolet radiation to human skin), benign prostatic hyperplasia, certain microbial infections including adenovirus, hantavirus, Borrelia burgdorferi, Yersinia spp., and Bordetella pertussis, thrombosis caused by platelet aggregation, reproductive disorders (such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menorrhagia), acute and chronic kidney diseases (including proliferative glomerulonephritis), hypertrophic scarring, endotoxic shock and fungal infections, familial adenomatous polyposis, myelodysplastic syndrome, aplastic anemia, ischemic injury, fibrosis of the lungs, kidneys, or liver, infantile hypertrophic pyloric stenosis, urinary tract obstruction syndrome, and psoriatic arthritis.

[1211] Methods of administration of such antibody conjugates include, but are not limited to, parenteral (e.g., intravenous) administration, such as, by bolus injection or continuous infusion over a period of time, oral administration, intramuscular administration, intratumoral administration, intramuscular administration, intraperitoneal administration, intracerebrospinal administration, subcutaneous administration, intra-articular administration, intrasynovial administration, lymph node injection, or intrathecal administration.

[1212] For the purpose of treating disease, the appropriate dosage of the antibody conjugate of the present invention depends on various factors, such as the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, prior therapies, and the patient's clinical history. The antibody conjugate can be administered once or as part of a series of treatments, lasting from several days to several months, or until a cure or a reduction in the disease state is achieved (e.g., tumor size reduction). The optimal dosing regimen can be calculated from cumulative drug measurements in the patient and will vary depending on the relative potency of the specific antibody conjugate. In some embodiments, the dosage is from 0.01 mg to 20 mg (e.g., 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg) / kg body weight, and may be administered once or more daily, weekly, monthly, or annually. In some embodiments, the antibody conjugate of the present invention is administered once every two weeks or once every three weeks. In some embodiments, the antibody conjugate of the present invention is administered only once. Therapists can estimate the repeat dosing rate based on the measured residence time and concentration of the drug in body fluids or tissues.

[1213] Pharmaceutical Composition

[1214] To prepare a pharmaceutical or sterile composition comprising one or more antibody conjugates described herein, the provided antibody conjugate may be mixed with a pharmaceutically acceptable carrier or excipient.

[1215] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY, 2000; Avis et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel). Marcel Dekker, New York, 1993; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, New York, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000.

[1216] In some embodiments, the pharmaceutical composition comprising the antibody conjugate of the present invention is a lyophilized formulation. In some embodiments, the pharmaceutical composition comprising the antibody conjugate is a lyophilized product containing the antibody conjugate, histidine, sucrose, and polysorbate 20 in a vial. In some embodiments, the pharmaceutical composition comprising the antibody conjugate is a lyophilized product containing the antibody conjugate, sodium succinate, and polysorbate 20 in a vial. In some embodiments, the pharmaceutical composition comprising the antibody conjugate is a lyophilized product containing the antibody conjugate, trehalose, citrate, and polysorbate 8 in a vial. The lyophilized product can be reconstituted, for example, with water or saline for injection. In a particular embodiment, the solution comprises the antibody conjugate, histidine, sucrose, and polysorbate 20 at a pH of about 5.0. In another particular embodiment, the solution comprises the antibody conjugate, sodium succinate, and polysorbate 20. In yet another particular embodiment, the solution comprises the antibody conjugate, dehydrated trehalose, dehydrated citrate, citric acid, and polysorbate 8 at a pH of about 6.6. For intravenous administration, the obtained solution is usually further diluted in a carrier solution.

[1217] The selection of an administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In some embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with acceptable levels of side effects. Therefore, the amount of biological product delivered depends in part on the specific entity and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, for example, Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY, 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY, 1993; Baert et al., New England Journal of Medicine 348:601-608, 2003; Milgrom et al., New... Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med. 344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619, 2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New Engl. J. Med. 343:1594-1602, 2000.

[1218] The appropriate dosage is determined by a clinician, for example, using parameters or factors known or suspected to affect or expected to affect treatment. Typically, the dosage begins slightly below the optimal dose and is then increased in small increments until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic measurements include those for symptoms (e.g., inflammation) or the levels of inflammatory cytokines produced.

[1219] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and route of administration without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the particular compound being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in the medical field.

[1220] Compositions containing the antibody conjugates of the present invention may be administered by continuous infusion or by doses at intervals such as daily, weekly, or 1-7 times per week, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks. Dosage may be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing regimens are those involving the maximum dose or frequency of administration to avoid significant undesirable side effects.

[1221] For the antibody conjugate of the present invention, the dose administered to the patient can be from 0.0001 mg / kg to 100 mg / kg of patient body weight. The dose can be between 0.001 mg / kg and 50 mg / kg of patient body weight, between 0.005 mg / kg and 20 mg / kg of patient body weight, between 0.01 mg / kg and 20 mg / kg of patient body weight, between 0.02 mg / kg and 10 mg / kg of patient body weight, between 0.05 mg / kg and 5 mg / kg of patient body weight, between 0.1 mg / kg and 10 mg / kg of patient body weight, between 0.1 mg / kg and 8 mg / kg of patient body weight, between 0.1 mg / kg and 5 mg / kg of patient body weight, between 0.1 mg / kg and 2 mg / kg of patient body weight, and between 0.1 mg / kg and 1 mg / kg of patient body weight. The dosage of the antibody conjugate can be calculated by multiplying the patient's weight in kilograms (kg) by the dose to be administered in mg / kg.

[1222] The dosage of the antibody conjugate of the present invention can be repeated, and each administration can be spaced less than 1 day, at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, 4 months, 5 months, or at least 6 months apart. In some embodiments, the antibody conjugate of the present invention is administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or at a lower frequency. In a particular embodiment, the dosage of the immunoconjugate of the present invention is repeated every 2 weeks.

[1223] The effective dose for a particular patient can vary depending on factors such as the condition to be treated, the patient’s overall health, the method, route and dosage of administration, and the severity of side effects (see, for example, Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, UrchPubl, London, 2001).

[1224] The route of administration can be by injection or infusion, for example, local or dermal application, subcutaneous, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional administration, or by continuous release systems or implants (see, for example, Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; L Anger, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; US Patent Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizer or a local anesthetic such as lidocaine for relieving injection site pain, or both. Alternatively, pulmonary administration may be used, for example, by using an inhaler or nebulizer, and formulations containing a nebulizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[1225] Examples of such other components are well known in the art.

[1226] Methods of co-administering or treating with a second therapeutic agent (e.g., cytokines, steroids, chemotherapy agents, antibiotics, or radiation) are known in the art (see, for example, Hardman et al., (ed.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson, (ed.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, PA; Chabner and Longo, (ed.) (2001) Cancer Chemotherapy and Biotherapy [Cancer Chemotherapy and Biotherapy], Lippincott, Williams & Wilkins [Lippincott, Williams & Wilkins Publishing Co., Philadelphia, PA]. An effective dose of the treatment can reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40%; or at least 50%.

[1227] Other therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with the antibody conjugate of the present invention may be administered at intervals of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, or about 8 hours to about 9 hours. The treatment may be administered at intervals of approximately 9 to 10 hours, approximately 10 to 11 hours, approximately 11 to 12 hours, approximately 12 to 18 hours, approximately 18 to 24 hours, approximately 24 to 36 hours, approximately 36 to 48 hours, approximately 48 to 52 hours, approximately 52 to 60 hours, approximately 60 to 72 hours, approximately 72 to 84 hours, approximately 84 to 96 hours, or approximately 96 to 120 hours. Two or more treatments may be administered during the same patient visit.

[1228] In some embodiments, the antibody conjugates of the present invention can be formulated to ensure appropriate in vivo distribution. Exemplary targeting portions include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988), Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. [European Federation of Biochemical Societies Letters] 357:140); Owais et al., (1995) Antimicrob. Agents Chemother. [Antimicrobial Chemotherapy] 39:180); Surfactant protein A receptor (Briscoe et al., (1995) Am.J. Physiol. [American Journal of Physiology] 1233:134); p120 (Schreier et al., (1994) J. Biol. Chem. [Journal of Biochemistry] 269:9090); See also K. Keinanen; ML Laukkanen (1994) FEBS Lett. [European Federation of Biochemical Societies Letters] 346:123); JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[1229] This invention provides a method for administering, alone or in combination with other therapies, a pharmaceutical composition comprising an antibody conjugate of the invention to a subject in need. The therapies of the combination therapies of the invention (e.g., prophylactic or therapeutic agents) can be administered simultaneously or sequentially to the subject. Therapies of the combination therapies of the invention (e.g., prophylactic or therapeutic agents) can also be cyclically administered. Cyclic therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., the cycle, to reduce resistance to a therapy (e.g., an agent), to avoid or reduce the side effects of a therapy (e.g., an agent), and / or to improve the efficacy of the therapy.

[1230] The combination therapies of the present invention (e.g., preventative or therapeutic agents) can be administered to subjects in parallel.

[1231] The term "parallel" is not limited to administering a therapy (e.g., a prophylactic or therapeutic agent) at exactly the same time, but rather means administering a pharmaceutical composition comprising the antibody of the present invention or a fragment thereof to a subject in such a sequence and at such time intervals that the antibody or antibody conjugate of the present invention can work in conjunction with one or more other therapies to provide increased benefit compared to if they were administered separately. For example, each therapy may be administered to a subject at the same time or sequentially at different time points in any order; however, if not administered at the same time, they should be administered sufficiently close in time to provide the desired therapeutic or preventative effect. Each therapy may be administered to a subject separately in any suitable form and via any suitable route. In various embodiments, therapies (e.g., preventative or therapeutic agents) are administered to the subject at intervals of less than 5 minutes, less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, 24 hours, 48 ​​hours, 72 hours, or 1 week. In other embodiments, two or more therapies (e.g., preventative or therapeutic agents) are administered during the same patient visit.

[1232] The prophylactic or therapeutic agent of a combination therapy may be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent of a combination therapy may be administered to the subject in parallel in separate pharmaceutical compositions. The prophylactic or therapeutic agent may be administered to the subject via the same or different routes of administration.

[1233] Example

[1234] The invention is further described in the following examples, which are not intended to limit the scope of the invention as described in the claims.

[1235] Temperatures are given in degrees Celsius. Unless otherwise specified, all evaporation is carried out under reduced pressure, typically between approximately 15 mm Hg and 100 mm Hg (=20–133 mbar). The structures of the final products, intermediates, and starting materials are confirmed by standard analytical methods (e.g., trace analysis and spectroscopic characterization (e.g., MS, IR, NMR)). Abbreviations used are those conventional in the art.

[1236] All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of this invention are commercially available or can be produced by organic synthesis methods known to those skilled in the art or by organic synthesis methods as described herein.

[1237] abbreviation:

[1238] The abbreviations used are those commonly used in this field or the following abbreviations:

[1239]

[1240]

[1241] Analytical methods

[1242] Acquire LC / MS data using an instrument with the following parameters:

[1243]

[1244] The method for generating LC / MS data is as follows:

[1245] Method A: 2-minute acid method

[1246]

[1247]

[1248] gradient:

[1249] Method B: 2min alkaline method

[1250]

[1251] gradient:

[1252] Method C: 5min acid method

[1253]

[1254] gradient:

[1255] Use an instrument with the following parameters to acquire HRMS data:

[1256]

[1257] The method for generating HRMS data for joints / payloads and synthetic intermediates is as follows:

[1258] Method D: 5min acid method

[1259]

[1260] gradient:

[1261] The method used to generate HRMS data for antibody-drug conjugates is as follows:

[1262] Method E: Protein methods

[1263]

[1264] gradient:

[1265] Obtained using an instrument with the following parameters and a 12-minute run time. Size exclusion chromatography data:

[1266]

[1267] Example 1: Synthesis of joint intermediates

[1268] Example 1-1: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (LI-1)

[1269]

[1270] Step 1: 2-(bromomethyl)-4-nitrobenzoic acid Synthesis

[1271] NBS (300.93 mg, 1.6908 mol) and AIBN (37.86 mg, 0.2305 mmol) were added to a stirred solution of 2-methyl-4-nitrobenzoic acid (300 g, 1.5371 mol) in CCl4 (3000 mL) at room temperature. The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was monitored by TLC analysis. The reaction mixture was diluted with saturated NaHCO3 solution (2 lit) and extracted with ethyl acetate (2 x 2 lit). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a 2%–3% ethyl acetate solution in petroleum ether as eluent to give 2-(bromomethyl)-4-nitrobenzoic acid (250 g, 59% yield). 1 H NMR (400MHz, CDCl3): δ8.35 (d, J=2.0Hz, 1H), 8.20 (q, J=8.8, 2.4Hz, 1H), 8.12 (d, J=8.8Hz, 1H), 4.97 (s, 2H), 4.00 (s, 3H).

[1272] Step 2: 4-Nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid Synthesis

[1273] At room temperature, propan-2-yn-1-ol (255.68 g, 265.50 mL, 4.5609 mol, d = 0.963 g / mL) and Cs₂CO₃ (743.03 g, 2.2805 mol) were added to a mixture of 2-(bromomethyl)-4-nitrobenzoic acid (250 g, 0.9122 mol) in ACN (5000 mL). The resulting mixture was heated to 80 °C and held for 16 h. The reaction mixture was filtered through a diatomaceous earth mat and washed with ethyl acetate (2 lit). The filtrate was concentrated under reduced pressure. A saturated NaHCO₃ solution (1 lit) was added to the obtained crude compound, and the aqueous layer was acidified to pH 2 using 2N HCl (2 lit). After filtration and vacuum drying, 4-nitro-2-((propan-2-yn-1-yloxy)methyl)benzoic acid (130 g, 60.6%) was obtained. 1 H NMR (400MHz, DMSO): δ13.61(brs,1H),8.37(d,J=2.4Hz,1H),8.23(dd,J=2.4,8.4Hz, 1H),8.10(d,J=8.8Hz,1H),4.95(s,2H),4.37(d,J=2.4Hz,2H),3.52(t,J=2.4Hz,1H)

[1274] Step 3: Methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate Synthesis

[1275] SOCl2 (526.08 g, 320.78 mL, 4.4219 mol, d = 1.64 g / mL) was slowly added to a stirred solution of 4-nitro-2-((propan-2-yn-1-yloxy)methyl)benzoic acid (130 g, 0.5527 mol) in MeOH (1300 mL) at 0 °C. The reaction was stirred at 70 °C for 4 h. The reaction solvent was evaporated under reduced pressure. The resulting residue was dissolved in ethyl acetate (1000 mL) and washed with saturated NaHCO3 (600 mL), water (500 mL), and a brine solution (500 mL). The separated organic layer was dried over sodium sulfate, filtered, and evaporated under reduced pressure to obtain methyl 4-nitro-2-((propan-2-yn-1-yloxy)methyl)benzoate (110 g, 80% yield). 1 H NMR (400MHz, CDCl3): δ8.56 (t, J = 0.8 Hz, 1H), 8.18-8.09 (m, 2H), 5.03 (s, 2H), 4.35 (d, J = 2.4Hz, 2H), 3.96 (s, 3H), 2.49 (t, J = 2.4Hz, 1H).

[1276] Step 4: Methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate Synthesis

[1277] Fe powder (197.21 g, 3.5310 mol) and NH4Cl (188.88 g, 3.5310 mol) were added to a solution of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate (110 g, 0.4414 mol) in a mixture of EtOH (1100 mL) and H2O (550 mL) at room temperature. The resulting mixture was heated at 80 °C for 16 h. The reaction mixture was cooled to room temperature and filtered through diatomaceous earth, and washed with ethyl acetate (2 lit). The filtrate was concentrated under reduced pressure to up to half its volume. Ethyl acetate (1.5 lit) was added to the residue and the two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 lit). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. Methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate (70 g, 72% yield) was purified by SiO2 column chromatography (15%-20% ethyl acetate in petroleum ether solution). 1HNMR (400MHz, CDCl3): δ7.67(d,J=8.8Hz,1H),6.78(t,J=1.6Hz,1H),6.48(q,J=8.4,2.4 Hz, 1H), 4.79 (s, 2H), 4.25 (d, J = 2.4Hz, 2H), 3.70 (d, J = 4.0Hz, 3H), 3.42 (t, J = 2.4Hz, 1H).

[1278] Step 5: (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol Synthesis

[1279] LiAlH4 (1M, in THF) (21.23 g, 798.2 mmol, 798.2 mL) was slowly added to a stirred solution of THF (1000 mL) at 0 °C. A solution of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate (70 g, 319.3 mmol) in THF (800 mL) was slowly added at 0 °C. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was cooled to 0 °C, and then water (22 mL) was added very slowly, followed by 20% NaOH (22 mL) and water (66 mL). The reaction mixture was stirred at 0 °C for 30 min. Anhydrous sodium sulfate was added to absorb excess water. The mixture was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate (1000 mL) and 10% MeOH / DCM (500 mL). The filtrate was concentrated under reduced pressure. The crude compound was purified by SiO2 column chromatography (using 35%-40% ethyl acetate in petroleum ether as eluent) to give (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (50.6 g, 83% yield). 1 H NMR (400MHz, CDCl3): δ6.98(d,J=8.0Hz,1H),6.56(d,J=2.4Hz,1H),6.43(dd,J=2.4,8.0Hz,1H),4.98(s,2 H), 4.64 (t, J = 5.2Hz, 1H), 4.47 (s, 2H), 4.34 (d, J = 5.6Hz, 2H), 4.15 (d, J = 2.4Hz, 2H), 3.46 (t, J = 2.4Hz, 1H).

[1280] Step 6: Synthesis of (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)carbamate

[1281]

[1282] N was added to a solution of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (1.92 g, 10.04 mmol, 1.0 equivalent), (9H-fluorene-9-yl)methyl(S)-(1-amino-1-oxo-5-ureidopent-2-yl)carbamate (3.99 g, 10.04 mmol, 1.0 equivalent), and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate (4.20 g, 11.04 mmol, 1.1 equivalent) in DMF (10 mL). N-Diisopropylethylamine (2.62 mL, 15.06 mmol, 1.5 equivalents). After stirring at ambient temperature for 1 hour, the mixture was poured into water (200 mL). The resulting solid was filtered, washed with water, and dried under vacuum to obtain (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (6.08 g, 99%). LCMS: MH+ = 571.5; Rt = 0.93 min (2 min acid method - Method A).

[1283] Step 7: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide

[1284]

[1285] Dimethylamine (2M, in THF, 21.31 mL, 42.62 mmol, 4 equivalents) was added to (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (6.08 g, 10.65 mmol, 1.0 equivalent). After stirring at ambient temperature for 1.5 hours, the supernatant was decanted from the formed gel-like residue. The residue was ground with diethyl ether (3 × 50 mL), and the resulting solid was filtered, washed with diethyl ether, and dried under vacuum. (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide (3.50 g, 10.04 mmol, 94%) was obtained. LCMS: MH+349.3; Rt=0.42min (2min acid method - Method A).

[1286] Step 8: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (LI-1)

[1287]

[1288] N,N-diisopropylethylamine (3.50 mL, 10.04 mmol, 1.0 equivalent) was added to a solution of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide (3.50 g, 10.04 mmol, 1.0 equivalent), (tert-butoxycarbonyl)-L-valine (2.62 g, 12.05 mmol, 1.2 equivalent), and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate (4.58 g, 12.05 mmol, 1.2 equivalent) in DMF (10 mL). After stirring at ambient temperature for 2 hours, the mixture was poured into water (200 mL), and the resulting suspension was precipitated with EtOAc (3 x 100 mL). Extracted by (mL). The combined organic layers were dried over sodium sulfate and concentrated under vacuum. Purified by ISCO SiO2 chromatography (0-20% methanol / dichloromethane), tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (LI-1) (2.49 g, 4.55 mmol, 45%). 1H NMR (400MHz, DMSO-d6) δ10.00(s,1H),7.96(d,J=7.7Hz,1H),7.55(dq,J=4.9,2.2Hz,2H,aryl),7.32(d,J=8.9Hz,1H,aryl),6.76(d,J=8.9Hz,1H),5.9 5(t,J=5.8Hz,1H),5.38(s,2H),5.01(t,J=5.5Hz,1H),4.54(s,2H),4.45(d d,J=25.2,5.3Hz,3H),4.20(d,J=2.4Hz,2H),3.83(dd,J=8.9,6.7Hz,1H),3. 49 (t, J = 2.4 Hz, 1H), 2.97 (dh, J = 26.0, 6.5 Hz, 2H), 1.96 (h, J = 6.6 Hz, 1H), 1.74-1.50 (m, 2H), 1.39 (m, 11H), 0.84 (dd, J = 16.2, 6.7 Hz, 6H). LCMS: MNa + 570.5; Rt = 0.79 min (2 min acidic method - Method A). Examples 1-2: Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (LI-2).

[1289]

[1290] Step 1: Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid

[1291]

[1292] Add 28.19 g, 502.43 mmol, 1.00 equivalent of KOH in 150 mL of H₂O to a solution of 90 g, 502.43 mmol, 1.00 equivalent of 6-nitroisobenzofuran-1(3H)-one in MeOH (1000 mL). Stir the brown mixture at 25 °C for 1.5 h. Concentrate the brown mixture under reduced pressure to give a residue and dissolve it in DCM (2000 mL). Add TBDPSCl (296.91 g, 1.08 mol, 277.49 mL, 2.15 equivalent of) and imidazole (171.03 g, 2.51 mol, 5.00 equivalent of) to the mixture and stir at 25 °C for 12 h. Concentrate the mixture under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 1 / 1) to give 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (34 g, 74.16 mmol, 14.76% yield) as a white solid. 1 H NMR(400MHz, methanol-d4)δppm1.13(s,9H)5.26(s,2H)7.34-7.48(m,6H)7.68(br d,J=8Hz,4H)8.24(br d,J=8Hz,1H)8.46(br d,J=8Hz,1H)8.74(s,1H)

[1293] Step 2: Synthesis of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol

[1294]

[1295] Add BH3·THF (1M, 470.68mL, 5 equivalents) to a mixture of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (41 g, 94.14 mmol, 1 equivalent) in THF (205 mL). Stir the yellow mixture at 60 °C for 2 h. Add MeOH (400 mL) to the mixture and concentrate under reduced pressure to give the residue. Then add H2O (200 mL) and DCM (300 mL), extract with DCM (3 × 200 mL), wash with brine (300 mL), dry to anhydrous MgSO4, filter, and concentrate under reduced pressure to give the residue. Purify the residue by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 1 / 1). (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol (34 g, 80.65 mmol, 85.7% yield) was obtained as a white solid.

[1296] 1H NMR (400MHz, methanol-d4) δppm 1.10 (s, 9H) 4.58 (s, 2H) 4.89 (s, 2H) 7.32-7.51 (m, 6H) 7.68 (dd, J=8, 1.38Hz, 4H) 7.76 (d, J=8Hz, 1H) 8.15 (dd, J=8 2.26Hz, 1H) 8.30 (d, J = 2Hz, 1H).

[1297] Step 3: Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde

[1298]

[1299] MnO2 (56.09 g, 645.22 mmol, 8 equivalents) was added to a solution of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol (34 g, 80.65 mmol, 1 equivalent) in DCM (450 mL). The black mixture was stirred at 25 °C for 36 h. MeOH (400 mL) was added to the mixture, and the mixture was concentrated under reduced pressure to give the residue. Then, H2O (200 mL) and DCM (300 mL) were added, and the mixture was extracted with DCM (3 × 200 mL), washed with brine (300 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel chromatography (CH2Cl2 = 100%). 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (30 g, 71.51 mmol, 88.7% yield) was obtained as a white solid.

[1300] 1H NMR (400MHz, chloroform-d) δppm 1.14(s,9H)5.26(s,2H)7.34-7.53(m,6H)7.60-7.73(m,4H)8.13(d,J=8Hz,1H)8.48(dd,J=8,2.51Hz,1H)8.67(d,J=2Hz,1H)10.16(s,1H)

[1301] Step 4: Synthesis of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine

[1302]

[1303] A solution of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (12.6 g, 30.03 mmol, 1 equivalent) in DCM (130 mL) was supplemented with propan-2-yn-1-amine (4.14 g, 75.08 mmol, 4.81 mL, 2.5 equivalents) and MgSO4 (36.15 g, 300.33 mmol, 10 equivalents), and the suspension mixture was stirred at 25 °C for 24 h. A small amount of the reaction solution was taken and treated with NaBH4, and TLC showed the formation of a new spot. The reaction mixture was filtered and concentrated under reduced pressure to give the residue. (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]propan-2-yn-1-imine (12 g, crude) was obtained as a yellow solid. 1 H NMR (400MHz, chloroform-d) δppm 1.11(s,9H)2.48(t,J=2.38Hz,1H)4.52(t,J=2.13Hz,2H)5.09(s,2H)7.35-7.49(m,6H)7.63-7.72(m, 4H) 7.79 (d, J = 8.53Hz, 1H) 8.25 (dd, J = 8.53, 2.51Hz, 1H) 8.68 (d, J = 2.26Hz, 1H) 8.84 (t, J = 1.88Hz, 1H).

[1304] (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-1-imine (12 g, 26.28 mmol, 1 equivalent) was dissolved in MeOH (100 mL) and THF (50 mL), then NaBH4 (1.49 g, 39.42 mmol, 1.5 equivalent) was added and the yellow mixture was stirred at -20 °C for 2 h. LCMS showed the detection of the desired compound. The reaction mixture was quenched by adding 200 mL of MeOH at -20 °C, and then concentrated under reduced pressure to give the residue. The residue was dissolved in 500 mL of EtOAc, washed with 150 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by rapid silica gel chromatography (eluent gradient of 0%-10% ethyl acetate / petroleum ether). N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 18.45 mmol, 70% yield) was obtained as a pale yellow oil. 1 H NMR (400MHz, chloroform-d) δppm 1.12(s,9H)2.13(t,J=2.38Hz,1H)3.33(d,J=2.51Hz,2H)3.80(s,2H)4.93(s,2H)7.36-7.49(m,6H)7.6 9(dd,J=7.91,1.38Hz,4H)7.77(d,J=8.53Hz,1H)8.16(dd,J=8.41,2.38Hz,1H)8.24(d,J=2.26Hz,1H).

[1305] Step 5: Synthesis of (9H-fluorene-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate

[1306]

[1307] A solution of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 19.62 mmol, 1 equivalent) and Fmoc-OSU (7.28 g, 21.59 mmol, 1.1 equivalent) in dioxane (90 mL) was added to saturated NaHCO3 (90 mL), and the white suspension was stirred at 20 °C for 12 h. The reaction mixture was diluted with 150 mL of H2O and extracted twice with EtOAc (150 mL each time). The combined organic layers were washed with 200 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by rapid silica gel chromatography (eluent: 0%–30% ethyl acetate / petroleum ether). A white solid (9H-fluorene-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate (7.7 g, 11.08 mmol, 56.48% yield, 98% purity) was obtained.

[1308] ¹H NMR (400MHz, chloroform-d) δppm 1.12 (s, 9H) 2.17 (br d, J = 14.31Hz, 1H) 3.87-4.97 (m, 9H) 6.98-8.28 (m, 21H).

[1309] Step 6: Synthesis of (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1310]

[1311] Zn (7.20 g, 110 mmol, 15 equivalences) was added to an ice-bath-cooled solution of (9H-fluorene-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate (5.0 g, 7.34 mmol, 1.0 equivalences) in 10% AcOH / CH2Cl2 (100 mL). The ice bath was removed, and the resulting mixture was stirred for 2 hours, during which time it was filtered through a diatomaceous earth mat. Volatile substances were removed under vacuum, and the residue was dissolved in EtOAc, washed with saturated NaHCO3 and NaCl, dried over MgSO4, filtered, concentrated, and subjected to ISCO SiO2 chromatography (0%-75% EtOAc / heptane) to obtain (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.99 g, 62%). LCMS: MH+ = 651.6; Rt = 3.77 min (5 min acidic method - Method C).

[1312] Step 7: Synthesis of (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1313]

[1314] Ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.27 g, 9.18 mmol, 2.0 equivalent) was added to (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.99 g, 4.59 mmol, 1.0 equivalent) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureidovalerate (1.72 g, 4.59 mmol, 1.0 equivalent) in CH2Cl2 (40 mL). After stirring for 10 minutes, MeOH (1 mL) was added to homogenize the solution. The reaction was stirred for 16 hours, volatiles were removed under vacuum, and the product was purified by ISCO SiO2 chromatography (0%-15% MeOH / CH2Cl2) to obtain (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.78 g, 60%). LCMS: MH+ = 1008.8; Rt = 3.77 min (5 min acidic method - Method C).

[1315] Step 8: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1316]

[1317] 2M dimethylamine in MeOH (30 mL, 60 mmol, 37 equivalences) and THF (10 mL) was added to (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (1.60 g, 1.588 mmol, 1.0 equivalences). After standing for 3 hours, the volatiles were removed under vacuum and the residue was ground with Et2O to remove Fmoc deprotection byproducts. CH2Cl2 (16 mL) and pyridine (4 mL) were added to the resulting solid, and propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equivalences) was added to the heterogeneous solution. After stirring for 30 min, additional propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equivalent) was added. After stirring for another 20 min, MeOH (1 mL) was added to quench the remaining chloroformate, and the volatiles were removed under vacuum. Purification by ISCO SiO2 chromatography (0%–15% MeOH / CH2Cl2) yielded propargyl-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(propargyl-1-yl)carbamate (984 mg, 71%). LCMS: MH+ = 867.8; Rt = 3.40 min (5 min acidic method – Method C).

[1318] Step 9: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (LI-2)

[1319]

[1320] Add 1.0 M tetrabutylammonium fluoride (2.27 mL, 2.27 mmol, 2.0 equivalent) in THF to a solution of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (984 mg, 1.135 mmol, 1.0 equivalent) in THF (7.5 mL). After standing for 6 hours, volatiles were removed under vacuum, and the residue was purified by ISCOSiO2 chromatography (0%-40% MeOH / CH2Cl2) to obtain propargyl-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(propargyl-1-yl)carbamate (629 mg, 88%). LCMS: MH+ = 629.6; Rt = 1.74 min (5 min acid method - Method C).

[1321] Examples 1-3: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3)

[1322]

[1323] Step 1: Synthesis of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide

[1324]

[1325] MeNH2 (3.00 kg, 29.94 mol, 600 mL, 31.0% purity) was added to a stirred suspension of 6-nitroisobenzofuran-1(3H)-one (500 g, 2.79 mol) in MeOH (1500 mL) at 25 °C and stirred for 1 h. The solid was filtered and washed twice with water (600 mL), and dried under high vacuum to obtain the residue. The product 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, crude product) was obtained as a white solid. LCMS: RT=0.537min, MS m / z=193.2. 1H NMR: 400MHz DMSOδ8.57(br d,J=4.4Hz,1H),8.31(dd,J=2.4,8.6Hz,1H),8.21(d,J=2.4Hz,1H),7.86(d,J= 8.8Hz, 1H), 5.54 (t, J = 5.6Hz, 1H), 4.72 (d, J = 5.5Hz, 2H), 2.78 (d, J = 4.4Hz, 3H).

[1326] Step 2: Synthesis of (2-((methylamino)methyl)-4-nitrophenyl)methanol

[1327]

[1328] A solution of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, 2.66 mol) in THF (5000 mL) was cooled to 0 °C, and then BH3-Me2S (506 g, 6.66 mol) (2.0 M, in THF) was added dropwise over 60 min, and the mixture was heated to 70 °C and held for 5 h. LCMS showed that the starting material was consumed. After completion, 4 M HCl (1200 mL) in methanol was added to the reaction mixture at 0 °C, and the mixture was heated at 65 °C for 8 h. The reaction mixture was cooled to 0 °C, the solid was filtered, and concentrated under reduced pressure. 520 g of (2-((methylamino)methyl)-4-nitrophenyl)methanol was obtained as a white solid. LCMS: RT = 0.742 min, MS m / z = 197.1 [M+H]+. 1 H NMR: 400MHz DMSO δ9.25(br s,2H),8.37(d,J=2.4Hz,1H),8.14(dd,J=2.4,8.5Hz,1H),7.63(d,J=8.4Hz,1H),5.72(br s,1H),4.65(s,2H),4.15(br s,2H),2.55-2.45(m,3H)

[1329] Step 3: Synthesis of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine

[1330]

[1331] A solution of (2-((methylamino)methyl)-4-nitrophenyl)methanol (520 g, 2.65 mol) and imidazole (721 g, 10.6 mol) in DCM (2600 mL) was cooled to 0 °C, and then TBDPS-Cl (1.09 kg, 3.98 mol, 1.02 L) was added dropwise while stirring the mixture for 2 h. The mixture was poured into ice-cold water (1000 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated under vacuum to give the crude product. The crude product was purified by silica gel chromatography (eluting with ethyl acetate: petroleum ether (from 10 / 1 to 1)) to give the residue. 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (600 g) was obtained as a yellow liquid. LCMS: Products: RT = 0.910 min, MS m / z = 435.2 [M+H]+

[1332] 1H NMR:400MHz CDCl3δ8.23(d,J=2.4Hz,1H),8.15(dd,J=2.4,8.4Hz,1H),7.76(d,J=8.4Hz,1H),7.7 1-7.66(m,4H),7.50-7.37(m,6H),4.88(s,2H),3.65(s,2H),2.39(s,3H),1.12(s,9H)

[1333] Step 4: Synthesis of (9H-fluorene-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate

[1334]

[1335] Fmoc-OSU (341.5 g, 1.01 mol) and Et3N (186.2 g, 1.84 mol, 256.2 mL) were added to a solution of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (400 g, 920.3 mmol) in THF (4000 mL), and the mixture was stirred at 25 °C for 1 h. The mixture was poured into water (1600 mL) and extracted twice with ethyl acetate (1000 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated under vacuum to give the crude product. The crude product was purified by silica gel chromatography (eluting with petroleum ether:ethyl acetate (from 1 / 0 to 1 / 1)) to give (9H-fluorene-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (405 g) as a white solid. LCMS: RT = 0.931 min, MS m / z = 657.2 [M+H]+.

[1336] 1H NMR: 400MHz CDCl3δ8.21-7.96(m,1H),7.87-7.68(m,3H),7.68-7.62(m,4H),7.62-7.47(m,2H),7.47-7.28(m,9H),7.26-7.05(m,2H),4.81(br s,1H),4.62-4.37(m,4H),4.31-4.19(m,1H),4.08-3.95(m,1H),2.87(br d,J=5.2Hz,3H),1.12(s,9H).

[1337] Step 5: Synthesis of (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1338]

[1339] A solution of (9H-fluorene-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (3.0 g, 4.57 mmol, 1.0 equivalent) in MeOH (90 mL) and EtOAc (30 mL) was degassed and purged into a balloon of N2 via a three-way stopcock. After repeating the degassed / N2 purging twice, 10% Pd / C deGussa type (0.486 g, 0.457 mmol, 0.1 equivalent) was added. The resulting mixture was degassed and purged into a balloon of 2H2 via a three-way stopcock. After repeating the degassed / H2 purging twice, the reaction was stirred under H2 balloon pressure for 4 hours. The reaction mixture was degassed and purged into N2, filtered through a diatomaceous earth pad, and further eluted with MeOH. After removing volatiles under vacuum and then aspirating under high vacuum, (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.78 g, 97%) was obtained. LCMS: MH+ = 627.7; Rt = 1.59 min (2 min acidic method - Method A). 1 H NMR: 400MHz CDCl3δ7.80(br d,J=7.2Hz,1H),7.74-7.67(m,5H),7.64(br d,J=6.8Hz,1H),7.49-7.30(m,10H),7.23-7.06(m,2H),6.61-6.41(m,2H),4.66(br d,J=7.2Hz,2H),4.55(s,2H),4.51-4.34(m,2H),4.32-4.10(m,1H),3.66(br s,2H),2.96-2.78(m,3H),,1.07(s,9H).

[1340] Step 6: Synthesis of (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1341]

[1342] Add 2-ethoxyquinoline-1(2H)-carboxylic acid ethyl ester (2.256 g, 9.12 mmol, 2.0 equivalent) to (9H-fluorene-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.86 g, 4.56 mmol, 1.0 equivalent) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureidovalerate (1.71 g, 4.56 mmol, 1.0 equivalent) to (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureidovalerate (1.71 g, 4.56 mmol, 1.0 equivalent) in 2:1CH2Cl2 / MeOH (60 mL). The homogenized solution was stirred for 16 hours, during which time additional (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureidovalerate (0.340 g, 0.2 equivalents) and ethyl 2-ethoxyquinoline-1(2H)-carboxylate (0.452 g, 0.4 equivalents) were added to drive the reaction to completion. After stirring for another 5 hours, the volatiles were removed under vacuum, and the solution was purified by ISCO SiO2 chromatography (0-5% MeOH / CH2Cl2) to obtain (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureidovalerate)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.95 g, 65%). LCMS: MH+ = 984.1; Rt = 1.54 min (2 min acid method - Method A).

[1343] Step 7: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1344]

[1345] Add 2.0 M dimethylamine in MeOH (10.42 mL, 20.85 mmol, 10 equivalences) to (9H-fluorene-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.05 g, 2.085 mmol, 1.0 equivalences). After stirring for 16 hours, remove volatiles under vacuum. Dissolve the residue in CH2Cl2 (20 mL) and add DIEA (0.533 mL, 4.17 mmol, 2 equivalences) and propargyl chloroformate (0.264 mL, 2.71 mmol, 1.3 equivalences). After stirring at room temperature for 16 hours, the reaction mixture was diluted with 20 mL of CH₂Cl₂, washed with saturated NaHCO₃ and NaCl, dried over MgSO₄, filtered, concentrated, and purified by ISCO SiO₂ chromatography (0%–15% MeOH / CH₂Cl₂) to give propan-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.04 g, 59%). LCMS: MH⁺ = 843.8; Rt = 1.35 min (2 min acid method – Method A).

[1346] Step 8: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3)

[1347]

[1348] Add 1.0 M tetrabutylammonium fluoride (3.80 mL, 3.80 mmol, 2.0 equivalent) in THF to a solution of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.6 g, 1.90 mmol, 1.0 equivalent) in THF (10.0 mL) at 0 °C. After heating to room temperature and stirring for 16 hours, the volatiles were removed under vacuum. The residue was dissolved in EtOAc, washed with saturated NaHCO3 and NaCl, dried over MgSO4, filtered, concentrated, and purified by ISCO SiO2 chromatography (0%-30% MeOH / CH2Cl2) to give propan-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutamido)-5-ureopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3) (1.0 g, 87%). LCMS: MH+ = 605.7; Rt = 0.81 min (2 min acid method - Method A).

[1349] Examples 1-4: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-((((prop-2-yn-1-yloxy)carbonyl)amino)acetamyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (LI-4)

[1350]

[1351] Step 1: Synthesis of (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopent-2-yl)carbamate

[1352]

[1353] To a solution of (4-amino-2-nitrophenyl)methanol (10 g, 59.5 mmol, 1.0 equivalent), (9H-fluorene-9-yl)methyl(S)-(1-amino-1-oxo-5-ureidopentan-2-yl)carbamate (23.64 g, 59.5 mmol, 1.0 equivalent), and 1-hydroxy-7-azabenzotriazole (8.50 g, 62.4 mmol, 1.05 equivalent) in DMF (50 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (11.97 g, 62.4 mmol, 1.05 equivalent) was added. After stirring at ambient temperature for 16 hours, the mixture was poured into water (4 L) and stirred for 30 minutes. The resulting solid was filtered, washed with water, and dried under vacuum. (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopent-2-yl)carbamate (31.49 g, 57.5 mmol, 97%) was obtained. LCMS: MH+ = 548; Rt = 2.02 min (5 min acid method - Method C).

[1354] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide

[1355]

[1356] Dimethylamine (2M, in MeOH, 331 mL, 661 mmol, 11.5 equivalence) was added to a solution of (9H-fluorene-9-yl)methyl(S)-(1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (31.49 g, 57.5 mmol, 1.0 equivalence) in DMF (50 mL). After stirring at ambient temperature for 24 hours, the volatiles were removed under vacuum, and the resulting residue was ground with diethyl ether (3 × 2 L). The residue was dried under vacuum to obtain (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide (21.85 g, 57.5 mmol, 99%). LCMS: MH+ = 326.4; Rt = 0.35 min (2 min acidic method - Method A).

[1357] Step 3: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[1358]

[1359] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5.52 g, 28.8 mmol, 1.0 equivalent) was added to a solution of (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide (10.89 g, 28.8 mmol, 1.0 equivalent), (tert-butoxycarbonyl)-L-valine (6.25 g, 28.8 mmol, 1.0 equivalent), and 1-hydroxy-7-azabenzotriazole (3.92 g, 28.8 mmol, 1.0 equivalent) in DMF (40 mL). After stirring at ambient temperature for 24 hours, the mixture was added dropwise to water (2 L), stirred for 30 molecule, and cooled to 4 °C overnight. The mixture was saturated with NaCl, and the resulting solid was filtered off and dried under vacuum. tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (11.96 g, 22.8 mmol, 79%) was obtained. LCMS: MH+ = 525.4; Rt = 0.79 min (2 min acidic method - Method A).

[1360] Step 4: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[1361]

[1362] tert-butyldimethylchlorosilane (13.68 g, 90.76 mmol, 4.0 equivalent) was added to a suspension of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (11.96 g, 22.8 mmol, 1.0 equivalent) and imidazole (15.52 g, 228 mmol, 10 equivalent) in DMF (31 mL). The resulting mixture was stirred at ambient temperature for 48 h, followed by heating at 45 °C for 4 h. The mixture was then poured into water and stirred for 96 h. The solid was filtered, washed with water (2 × 100 mL), and dried under vacuum. After purification by SiO2 ISCO chromatography (0%-30% methanol / dichloromethane), tert-butyl((S)-1-(((S)-1-((4-((((tert-butyldimethylsilyl)oxy)methyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8.02 g, 12.56 mmol, 55%) was obtained. LCMS: MH+ = 639.6; Rt = 1.22 min (2 min acidic method - Method A).

[1363] Step 5: Synthesis of tert-butyl((S)-1-(((S)-1-((3-amino-4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[1364]

[1365] Under a nitrogen atmosphere, palladium on carbon (10 wt%, 2.00 g, 1.884 mmol, 0.15 equivalent) was added to a solution of tert-butyl((S)-1-(((S)-1-((4-((((tert-butyldimethylsilyl)oxy)methyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8.02 g, 12.56 mmol, 1.0 equivalent) in methanol (250 mL). The mixture was placed under 1 atm dihydrogen ionomer and stirred at ambient temperature for 18 hours. The mixture was filtered through diatomaceous earth and concentrated under vacuum. After purification by SiO2 ISCO chromatography (0%-40% methanol / dichloromethane), tert-butyl((S)-1-(((S)-1-((3-amino-4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (4.82 g, 7.92 mmol, 63%) was obtained. LCMS: MH+ = 609.6; Rt = 2.65 min (5 min acidic method - Method C).

[1366] Step 6: Synthesis of tert-butyl((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-((((prop-2-yn-1-yloxy)carbonyl)amino)acetamyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (LI-4)

[1367]

[1368] Step 6a):

[1369] Propyl chloroformate (5.0 g, 42.5 mmol, 1.0 equivalent) was added to a solution of glycine (3.19 g, 42.5 mmol, 1.0 equivalent) in 2 M aqueous sodium hydroxide solution (63.3 mL, 127 mmol NaOH, 3.0 equivalent). The resulting mixture was stirred at ambient temperature for 3 hours. The mixture was extracted with ethyl acetate (3 x 250 mL). The combined organic layers were dried over magnesium sulfate, filtered, and volatiles were removed under vacuum. After drying, ((prop-2-yn-1-yloxy)carbonyl)glycine was obtained. (3.97g, 25.3mmol, 59%). 1H NMR (400MHz, DMSO-d6) δppm 3.48 (t, J = 2.40Hz, 1H) 3.66 (d, J = 6.19Hz, 2H) 4.63 (d, J = 2.40Hz, 2H) 7.63 (t, J = 6.13Hz, 1H) 12.57 (br s, 1H).

[1370] Step 6b):

[1371] Add ((prop-2-yn-1-yloxy)carbonyl)glycine (0.732 g, 4.66 mmol, 1.05 equivalent), 1-hydroxy-7-azabenzotriazole (0.664 g, 4.88 mmol, 1.1 equivalent), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.935 g, 4.88 mmol, 1.1 equivalent) to a solution of tert-butyl((S)-1-(((S)-1-((3-amino-4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)a...

Claims

1. A compound having the structure of formula (II) or a pharmaceutically acceptable salt thereof, in: R 1 yes L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)NH((CH2) m O) t (CH2) n -**; *-C(=O)O(CH2) m C(=O)NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) n C(=O)-**; *-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**; *-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; Each R 3 Independently selected from H and C1-C6 alkyl; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; A is a bond or -OC(=O)-*, wherein the * of A indicates the point of attachment to D; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -CH2N(R b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、-NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, or -CH2N(R b )C(=O)CH2-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2 or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 Attachment point; and The * of L3 indicates that 2 attachment point; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R1 is L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 Attachment point; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2 or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 Attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; A is a bond or -OC(=O)-*, wherein the * of A indicates the point of attachment to D; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 1 yes L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 Attachment point; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; A is a bond or -OC(=O)-*, wherein the * of A indicates the point of attachment to D; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 1 yes L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, where the * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 1 Attachment point; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; A is a bond or -OC(=O)*, where * indicates the point of attachment to D; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 1 yes L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, where the * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 1 Attachment point; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; A is a bond or -OC(=O)*, where * indicates the point of attachment to D; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 1 yes L1 is *-C(=O)(CH2) m O(CH2) m -**, where the * of L1 indicates the attachment point with Lp and the ** of L1 indicates the connection point with R 1 Attachment point; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion of W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 It is polyethylene glycol; A is a bond or -OC(=O)*, where * indicates the point of attachment to D; and D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of the drug moiety.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

10. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

11. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

12. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

13. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

14. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure: wherein Xb is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

15. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

17. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

18. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

19. The compound of claim 1 or a pharmaceutically acceptable salt thereof, having the structure:

20. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

21. A linker having a structure of formula (VI), L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)NH((CH2) m O) t (CH2) n -**; *-C(=O)O(CH2) m C(=O)NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) n C(=O)-**; *-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**; *-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, where the * of L1 indicates the point of attachment to Lp; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; Each R 3 Independently selected from H and C1-C6 alkyl; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; A is a bond, -OC(=O)-; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -CH2N(R b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、-NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, or -CH2N(R b )C(=O)CH2-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2, or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 attachment point; and The * of L3 indicates that 2 attachment point.

22. The connector of claim 21, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * of L1 indicates the attachment point to Lp; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2 or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 Attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; and A is a bond or -OC(=O)-.

23. The connector of claim 21, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * of L1 indicates the attachment point to Lp; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; and A is a bond or -OC(=O)-.

24. The connector of claim 21, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * of L1 indicates the attachment point to Lp; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; and A is a bond or -OC(=O)-.

25. The connector of claim 21, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * of L1 indicates the attachment point to Lp; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; and A is a bond or -OC(=O)-.

26. The connector of claim 21, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -**, where the * of L1 indicates the point of attachment to Lp; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; L3 is a structure The spacer portion, in W is -CH2O-**, -CH2N(R b )C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR 2 )C(=O)O-**, or -C(=O)N(XR 2 )-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the 2 attachment point; and The * of L3 indicates that 2 Attachment point; R 2 is polyethylene glycol; and A is a bond or -OC(=O)-.

27. The joint of claim 21, having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

28. The joint of claim 21, having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

29. The joint of claim 21, having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

30. The connector of claim 21, having the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

31. The joint of claim 21, having the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

32. The connector of claim 21, having the structure: wherein Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

33. The joint of claim 21, having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

34. The joint of claim 21, having the structure: wherein Xb is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

35. The connector of claim 21, having the structure:

36. The connector of claim 21, having the structure:

37. The connector of claim 21, having the structure:

38. The connector of claim 21, having the structure:

39. The connector of claim 21, having the structure:

40. A conjugate having formula (IV): in: Ab is an antibody or a fragment thereof; R 100 yes Wherein R 100 The *** indicates the attachment point to the Ab; L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; -C(=O)NH((CH2) m O) t (CH2) n -**; *-C(=O)O(CH2) m C(=O)NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) m -**; *-C(=O)(CH2) m NH(CH2) n C(=O)-**; *-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n C(=O)NH(CH2) m -**; *-C(=O)(CH2) m C(R 3 )2-** or *-C(=O)(CH2) m C(=O)NH(CH2) m -**, where the * of L1 indicates the point of attachment to Lp; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; Each R 3 Independently selected from H and C1-C6 alkyl; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; A is a bond or -OC(=O)-; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -CH2N(R b )C(=O)O-**、-NHC(=O)C(R b )2NHC(=O)O-**, -NHC(=O)C(R b )2NH-**、-NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2N(XR 2 )C(=O)-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**、-CH2NR b C(=O)NR b -**, or -CH2N(R b )C(=O)CH2-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-*、***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*,***-(CH2CH2O) n -C(O)NHS(O)2NH-(CH2CH2O) n -*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2 or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 Attachment point; and The * of L3 indicates that 2 Attachment point; D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of said drug moiety, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

41. The conjugate of claim 40, wherein Ab is an antibody or a fragment thereof; R 100 yes Wherein R 100 The *** indicates the attachment point to the Ab; L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**; or *-C(=O)NH((CH2) m O) t (CH2) n -, wherein the * of L1 indicates the attachment point to Lp; R 2 is a hydrophilic moiety selected from polyethylene glycol and polyalkylene glycol; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each t is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a divalent peptide spacer wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group; A is a bond or -OC(=O)-; L3 is a structure The spacer portion, in (i) W is -CH2O-**, -NHC(=O)C(R b )2NHC(=O)-**、-CH2N(XR 2 )C(=O)O-**、-C(=O)N(XR 2 )-**、-CH2NR b C(=O)-**、-CH2NR b C(=O)NH-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is a bond or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 point of attachment; or (ii) W is -CH2O-** or -CH2NR b C(=O)-**, where each R b independently selected from H or C1-C6 alkyl and wherein the ** of W indicates the point of attachment to X; X is ***-C 4-6 Cycloalkylene-OC(O)NHS(O)2NH-*、***-(CH2CH2O) n -C(O)NHS(O)2NH-*, or ***-CH2-triazolyl-C 1-4 Alkylene-OC(O)NHS(O)2NH-(CH2CH2O) n -*, wherein each n is independently 1, 2 or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 Attachment point; and The * of L3 indicates that 2 Attachment point; D is a drug moiety comprising N or O, wherein D is attached to A via a direct bond from A to the N or O of said drug moiety, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

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