Antibody drug conjugates with linkers comprising hydrophilic groups

Abstract

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

Description

[0001] This application is a divisional application of Chinese patent application No. 202080036837.5, with an application date of May 19, 2020, a priority date of May 20, 2019, and an invention name of “Antibody drug conjugate with a linker containing a hydrophilic group”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 850,094, filed May 20, 2019, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0004] The present invention provides linkers for improving the solubility of antibody drug conjugates (ADCs) containing one or more hydrophobic drug compounds. Background Art

[0005] One aspect of antibody drug conjugate (ADC) design is the design of the chemical linker, which connects the drug moiety to the targeting moiety. Typically, ADCs use hydrophobic drug moieties, but when such drug moieties are used in combination with relatively hydrophobic linkers, solubility issues arise, which can affect the biocompatibility and drug efficacy of the ADC.

[0006] Linker strategies have been reported that attempt to overcome these challenges, in particular hydrophilic linkers incorporating polyethylene glycol (see RP Lyon, TD Bovee, SO Doronina, PJ Burke, JH Hunter, HD Neff-LaFord, M. Jonas, ME Anderson, JR Setter, PD Senter, Nat. Biotechnol., 2015, 33, 733-735 and WO 2015057699), linkers incorporating sulfonates (RY Zhao, SD Wilhelm, C. Audette, G. Jones, BA Leece, A Lazar, VS Goldmacher, R. Singh, Y. Kovtun, WC Widdison, JM Lambert, RVJ Chari, J. Med. Chem., 2011, 54, 3606-3623) and linkers with carbohydrate backbones (FS Ekholm, H. A. Vilkman,V. J. Helin, J. Saarinen, T. Satomaa, Chem Med Chem. [Chem Med Chem., 2016, 11(22): 2501-2505). However, there is still a need for antibody drug conjugate formats that allow targeted delivery of hydrophobic drugs with improved pharmacokinetic and pharmacodynamic properties. Summary of the Invention

[0007] The present invention provides linkers for use in improving the solubility of linker-drug conjugates, wherein such conjugates comprise one or more hydrophobic drug compounds, wherein the linker comprises one or more hydrophilic groups. Various embodiments of the present invention are described herein.

[0008] The present invention further provides a linker for use in improving the solubility of an antibody drug conjugate (ADC), wherein the ADC comprises one or more hydrophobic drug compounds, wherein the linker comprises one or more hydrophilic groups. Various embodiments of the present invention are described herein.

[0009] In one embodiment, disclosed herein are linkers comprising one or more suicide groups, wherein each of the one or more suicide groups is substituted with one or more hydrophilic moieties.

[0010] In one embodiment, disclosed herein are linker-drug groups, wherein the linker comprises one or more suicide groups coupled to the drug, and wherein each of the one or more suicide groups is substituted with one or more hydrophilic moieties.

[0011] In one embodiment, disclosed herein are antibody drug conjugates comprising one or more linker-drug groups, wherein the linker comprises one or more suicide groups coupled to the drug, and wherein the one or more suicide groups are each substituted with one or more hydrophilic moieties.

[0012] In one embodiment, is a linker-drug group having formula (I), or a pharmaceutically acceptable salt thereof:

[0013]

[0014] in:

[0015] R 1 It is a reactive group;

[0016] L1 is a bridging spacer;

[0017] Lp is a divalent peptide spacer;

[0018] G-L2-A is a suicide spacer;

[0019] R2 It is the hydrophilic part;

[0020] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0021] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0022] L3 is the spacer portion;

[0023] and

[0024] 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.

[0025] In embodiments of linker-drug groups having formula (I), are linker-drug groups having formula (II), or a pharmaceutically acceptable salt thereof:

[0026]

[0027] in:

[0028] R 1 It is a reactive group;

[0029] L1 is a bridging spacer;

[0030] Lp is a bivalent peptide linker consisting of one to four amino acid residues;

[0031] R 2 It is the hydrophilic part;

[0032] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0033] L3 is the spacer portion;

[0034] and

[0035] 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.

[0036] In one embodiment, the antibody drug conjugate is of formula (III):

[0037]

[0038] in:

[0039] Ab is an antibody or its fragment;

[0040] R 100 is a coupling group;

[0041] L1 is a bridging spacer;

[0042] Lp is a divalent peptide linker;

[0043] G-L2-A is a suicide spacer;

[0044] R 2 It is the hydrophilic part;

[0045] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0046] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0047] L3 is the spacer portion;

[0048] 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,

[0049] and

[0050] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0051] In an embodiment of the antibody drug conjugate having formula (III) is an antibody drug conjugate having formula (IV):

[0052]

[0053] in:

[0054] Ab is an antibody or its fragment;

[0055] R 100 is a coupling group;

[0056] L1 is a bridging spacer;

[0057] Lp is a bivalent peptide linker consisting of one to four amino acid residues;

[0058] R 2 It is the hydrophilic part;

[0059] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0060] L3 is the spacer portion;

[0061] 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,

[0062] and

[0063] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0064] Another aspect of the invention is a linker having a structure of formula (V),

[0065]

[0066] in

[0067] L1 is a bridging spacer;

[0068] Lp is a divalent peptide spacer;

[0069] G-L2-A is a suicide spacer;

[0070] R 2 It is the hydrophilic part;

[0071] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0072] 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 and wherein the ** of A indicates the point of attachment to L2,

[0073] and

[0074] L3 is the spacer portion.

[0075] In an embodiment of a linker having formula (V), is a linker having a structure of formula (VI),

[0076]

[0077] in

[0078] L1 is a bridging spacer;

[0079] Lp is a divalent peptide spacer;

[0080] R 2 It is the hydrophilic part;

[0081] 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,

[0082] and

[0083] L3 is the spacer portion.

[0084] The linkers described herein comprise a hydrophilic portion that contributes to the overall hydrophilicity of the antibody-drug conjugate (ADC) and improves the water solubility of the ADC. The linkers described herein also unexpectedly reduce ADC aggregation and improve the pharmacokinetic and pharmacodynamic properties of the ADC. In addition, the hydrophilic linkers described herein allow for improved water solubility of the linker-drug groups described herein, thereby allowing for improved conjugation of antibodies to the linker-drug groups, which improves the purification and overall synthesis yield of ADCs (particularly ADCs comprising a hydrophobic drug portion). BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1Line graphs of cellular activity of antibody drug conjugates titrated in selected cell lines (A: HT-29PCAD+; B: FaDu; C: HCC70; D: HT-29; and E: HCC1954).

[0086] Figure 2 A: Line graph of caspase-3 / 7 activity of antibody drug conjugates titrated in HCC1954 cell line after 24 hours and B: 48 hours.

[0087] 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) Anti-tumor response; B) and C) Change in body weight compared to treatment initiation. Data are presented as mean ± SEM. *p < 0.05, compared to the untreated group at day 20 (one-way ANOVA with post hoc Dunnett's test). DETAILED DESCRIPTION

[0088] Various enumerated embodiments of the present invention are described herein. It should be appreciated that features specified within each embodiment can be combined with other specified features to provide further embodiments of the present invention.

[0089] Throughout this application, if there is a discrepancy between the specification text (eg, Table 3) and the sequence listing, the specification text controls.

[0090] definition

[0091] As used herein, term " alkyl " refers to the straight chain or branched hydrocarbon chain group being only composed of carbon and hydrogen atom, and there is no unsaturation in described group.As used herein, term " C1-C6 alkyl " refers to the straight chain or branched hydrocarbon chain group being only composed of carbon and hydrogen atom, and there is no unsaturation in described group, there is from one to six carbon atom, and is attached to the remainder of molecule by singly-bound.The limiting examples of " C1-C6 alkyl " group includes methyl (C1 alkyl), ethyl (C2 alkyl), 1- methylethyl (C3 alkyl), n-propyl (C3 alkyl), isopropyl (C3 alkyl), normal-butyl (C4 alkyl), isobutyl (C4 alkyl), sec-butyl (C4 alkyl), the tert-butyl (C4 alkyl), n-pentyl (C5 alkyl), isopentyl (C5 alkyl), neopentyl (C5 alkyl) and hexyl (C6 alkyl).

[0092] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, said radical containing at least one double bond. e"Alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, attached to the remainder of the molecule by a single bond. Non-limiting examples of "C2-C6 alkenyl" radicals include vinyl (C2 alkenyl), prop-1-enyl (C3 alkenyl), but-1-enyl (C4 alkenyl), pent-1-enyl (C5 alkenyl), pent-4-enyl (C5 alkenyl), pent-1,4-dienyl (C5 alkenyl), hex-1-enyl (C6 alkenyl), hex-2-enyl (C6 alkenyl), and 1,4-dienyl (C5 alkenyl). , hex-3-enyl (C6 alkenyl), hex-1-, 4-dienyl (C6 alkenyl), hex-1-, 5-dienyl (C6 alkenyl) and hex-2-, 4-dienyl (C6 alkenyl). As used herein, the term "C2-C3 alkenyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, the group containing at least one double bond, having from two to three carbon atoms, attached to the rest of the molecule by a single bond. Non-limiting examples of "C2-C3 alkenyl" groups include vinyl (C2 alkenyl) and prop-1-enyl (C3 alkenyl).

[0093] As used herein, term " alkylidene " refers to the divalent straight chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and there is no unsaturation in the group. As used herein, term " C1-C6 alkylidene " refers to the divalent straight chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and there is no unsaturation in the group, with from one to six carbon atoms. The limiting examples of " C1-C6 alkylidene " group includes methylene (C1 alkylidene), ethylidene (C2 alkylidene), 1-methylethylidene (C3 alkylidene), n-propylene (C3 alkylidene), isopropylidene (C3 alkylidene), n-butylidene (C4 alkylidene), isobutylidene (C4 alkylidene), secondary butylidene (C4 alkylidene), tert-butylidene (C4 alkylidene), n-pentylene (C5 alkylidene), isopentylene (C5 alkylidene), neopentylene (C5 alkylidene) and hexylene (C6 alkylidene).

[0094] As used herein, the term "alkenylene" refers to a divalent straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, and the group contains at least one double bond. As used herein, the term "C2-C6 alkenylene" refers to a divalent straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, the group contains at least one double bond, and has from two to six carbon atoms. Non-limiting examples of "C2-C6 alkenylene" groups include vinylene (C2 alkenylene), prop-1-enylene (C3 alkenylene), but-1-enylene (C4 alkenylene), pent-1-enylene (C5 alkenylene), pent-4-enylene (C5 alkenylene), pent-1,4-dienylene (C5 alkenylene), hex-1-enylene (C6 alkenylene), hex-2-enylene (C6 alkenylene), hex-3-enylene (C6 alkenylene), hex-1-,4-dienylene (C6 alkenylene), hex-1-,5-dienylene (C6 alkenylene) and hex-2-,4-dienylene (C6 alkenylene). As used herein, the term "C2-C6 alkenylene" refers to a divalent straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one double bond and having from two to three carbon atoms. Non-limiting examples of "C2-C3 alkenylene" groups include ethenylene (C2 alkenylene) and prop-1-enylene (C3 alkenylene).

[0095] 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.

[0096] As used herein, the term "polyethylene glycol" or "PEG" refers to a linear chain, branched chain, or star-shaped conformation composed of (OCH2CH2) groups. In certain embodiments, the polyethylene or PEG group is -(OCH2CH2) t *-, wherein t is 4-40, and wherein "-" indicates the end pointing toward the suicide spacer and "*-" indicates the point of attachment to the terminal group R', wherein R' is OH, OCH3, or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -(CH2CH2O) t *-, wherein t is 4-40, and wherein "-" indicates the end pointing toward the suicide spacer and "*-" indicates the point of attachment to the terminal group R", wherein R" is H, CH3 or CH2CH2C(=O)OH.

[0097] As used herein, the term "polyethylene glycol" refers to a m ) t In certain embodiments, the polyethylene or PEG group is -(O(CH2) m ) t *-, wherein m is 1-10, t is 4-40, and wherein "-" indicates the end pointing toward the suicide spacer and "*-" indicates the point of attachment to the terminal group R', wherein R' is OH, OCH3, or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -((CH2) m O) t *-, wherein m is 1-10, t is 4-40, and wherein "-" indicates the end pointing toward the suicide spacer and "*-" indicates the point of attachment to the terminal group R", wherein R" is H, CH3 or CH2CH2C(=O)OH.

[0098] As used herein, the term "drug moiety," "D," or "drug" refers to any compound having a desired biological activity and a reactive functional group that can be used to incorporate a drug into the linker-drug group of the present invention. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing a disease in a human or other animal. The reactive functional group forms a bond with "A" in the compounds of formula (I) and formula (II) and the conjugates of formula (III) and formula (IV). In some embodiments, the drug moiety has a nitrogen atom that can form a bond with "A." In other embodiments, the drug moiety has a hydroxyl group that can form a bond with "A." In other embodiments, the drug moiety has a carboxylic acid that can form a bond with "A." In other embodiments, the drug moiety has a carbonyl group that can form a bond with "A." In yet other embodiments, the drug moiety has a sulfhydryl group that can form a bond with "A."

[0099] If the required reactive functional groups are present, the term "drug moiety," "D," or "drug" further refers to a chemical that is recognized as a drug by the official United States Pharmacopeia, the official Homeopathic Pharmacopeia of the United States, or the official National Formulary, or any supplement thereof. 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).

[0100] In one embodiment, the drug moiety (D) can be a cytotoxic drug, a cytostatic drug or an immunosuppressive drug. Such cytotoxic drugs or immunosuppressive drugs include, for example, anti-tubulin agents, tubulin inhibitors, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of such cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycins, etoposide, maytansine and maytansinoids, taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]-benzodiazepines (PBD), indoline and benzodiazepines, and oxazolidinone and benzodiazepines) and vinca alkaloids.

[0101] In embodiments where the drug moiety is hydrophobic, the effects of the present invention will be more pronounced. Therefore, the drug moiety of the present invention is preferably hydrophobic and has an SlogP value of 1.5 or greater, 2.0 or greater, or 2.5 or greater. In some aspects, the drug used in the present invention has an 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.

[0102] Hydrophobicity can be measured using SlogP. SlogP is defined as the logarithm of the octanol / water partition coefficient (including implicit hydrogens) and can be calculated using the MOE from the Chemical Computing Group. TMThe program was used for calculation (SlogP values ​​were calculated using Wildman, 25 S.A., Crippen, GM; Prediction of Physiochemical Parameters by Atomic Contributions; J. Chern. Inf Comput. Sci. 39 No. 5 (1999) 868-873).

[0103] As used herein, the term "reactive group" is a functional group that is 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 of Table 1 provided herein.

[0104] As used herein, the term "coupling group" refers to a divalent moiety that connects a bridging spacer to an antibody or fragment thereof. The coupling group is a divalent moiety formed by reaction between a reactive group and a functional group of the antibody or fragment thereof. Non-limiting examples of such divalent moieties include the divalent chemical moieties given in Tables 1 and 2 provided herein.

[0105] As used herein, the term "bridging spacer" refers to one or more linker components that are covalently attached together to form a bivalent moiety that connects a bivalent peptide spacer to a reactive group or connects a bivalent peptide spacer to a coupling group. In certain embodiments, the "bridging spacer" comprises a carboxyl group attached to the N-terminus of the bivalent peptide spacer via an amide bond.

[0106] As used herein, the term "spacer moiety" refers to one or more linker components that are covalently attached together to form the moiety that connects the suicide spacer to the hydrophilic moiety.

[0107] As used herein, the term "bivalent peptide spacer" refers to a bivalent linker comprising one or more amino acid residues covalently attached together to form a portion connecting the bridging spacer to the suicide spacer. The one or more amino acid residues can be 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), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, and demethylpyrrolysine.

[0108] In certain embodiments, a "divalent peptide spacer" is a combination of two to four amino acid residues, wherein each residue is independently selected from a residue selected from the group consisting of 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), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, and demethylpyrrolysine, such as -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 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], where "-" indicates the point of attachment to the bridging spacer and "*" indicates the point of attachment to the suicide spacer.

[0109] As used herein, the term "linker component" refers to the following:

[0110] a) Alkylene group: -(CH2) n -, which may be linear or branched (in which case n is 1-18);

[0111] b) alkenylene groups;

[0112] c) an alkynylene group;

[0113] d) alkenyl groups;

[0114] e) an alkynyl group;

[0115] f) ethylene glycol unit: -OCH2CH2 or -CH2CH2O;

[0116] g) Polyethylene glycol unit: (-CH2CH2O-) x (where x is 2-20 in this case);

[0117] h)-O;

[0118] i)-S;

[0119] j) Carbonyl: -C(=O);

[0120] k) Ester: -C(=O)-O- or -OC(=O);

[0121] 1) Carbonate: -OC(=O)O;

[0122] m) amine: -NH;

[0123] n) Tertiary amine

[0124] o) Amide: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C 1-6 alkyl);

[0125] p) Carbamate: -OC(=O)NH- or -NHC(=O)O;

[0126] q) Urea: -NHC(=O)NH;

[0127] r) Sulfonamide: -S(O)2NH- or -NHS(O)2;

[0128] s) Ether: -CH2O- or -OCH2;

[0129] t) alkylene substituted with one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate, and phosphonate;

[0130] u) alkenylene substituted with one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate, and phosphonate;

[0131] v) alkynylene substituted with one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate, and phosphonate;

[0132] w)C1-C 10 alkylene groups in which one or more methylene groups are replaced by one or more -S-, -NH-, or -O- moieties;

[0133] x) a ring system having two available points of attachment, for example, a divalent ring selected from 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;

[0134] y) an amino acid residue selected from the group consisting of 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), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine, and demethylpyrrolysine;

[0135] A combination of two or more amino acid residues, wherein each residue is independently selected from the group consisting of 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), valeric acid (Nva), norleucine (Nle), selenocysteine ​​(Sec), pyrrolysine (Pyl), homoserine, homocysteine Acids and demethylpyrrolysine, 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;

[0136] as well as

[0137] z) a suicide spacer, wherein the suicide spacer comprises one or more protecting (triggering) groups that are 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

[0138] Non-limiting examples of such suicide spacers include:

[0139]

[0140] in:

[0141] PG is a protecting (triggering) group;

[0142] X a is O, NH or S;

[0143] X b is O, NH, NCH3 or S;

[0144] X c is O or NH;

[0145] Y a is CH2, CH2O or CH2NH;

[0146] Y b is CH2, O or NH;

[0147] Y c is a bond, CH2, O, or NH, and

[0148] LG is a leaving group, such as the drug portion (D) of the linker-drug group of the present invention.

[0149] Additional non-limiting examples of such suicide spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492-7509.

[0150] Additionally, the linker component can be a chemical moiety that is readily formed by a reaction between two reactive groups. Non-limiting examples of such chemical moieties are given in Table 1.

[0151] Table 1

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] Where: 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 by 1 to 3 -OH groups 1-4 Alkyl; each R in Table 1 7 Independently selected from H, C 1-6 Alkyl, fluorine, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C 1-4 Alkoxy and C substituted by -C(=O)OH 1-4 Alkyl; R in Table 1 37are independently selected from H, phenyl and pyridine; q in Table 1 is 0, 1, 2 or 3; R in Table 1 8 or R 13 is H or methyl; and R in Table 1 9 or R 14 is H, -CH3 or phenyl; and R in Table 1 is H or a suitable substituent, for example, an alkyl group.

[0159] Additionally, the linker component may be a group given in Table 2 below.

[0160] Table 2

[0161]

[0162]

[0163]

[0164]

[0165] As used herein, when showing a portion of the structure of a compound, a wavy line The point of attachment of the partial structure to the rest of the molecule is indicated.

[0166] As used herein, the term "suicide spacer" refers to a moiety comprising 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 that results in 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.

[0167] Non-limiting examples of such suicide spacers include:

[0168]

[0169] wherein such groups may be optionally substituted, and

[0170] in:

[0171] TG is the trigger group;

[0172] X a is O, NH or S;

[0173] X b is O, NH, NCH3 or S;

[0174] Xc is O or NH;

[0175] Y a is CH2, CH2O or CH2NH;

[0176] Y b is CH2, O or NH;

[0177] Y c is a bond, CH2, O, or NH, and

[0178] LG is a leaving group, such as the drug portion (D) of the linker-drug group of the present invention.

[0179] Additional non-limiting examples of such suicide spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492-7509.

[0180] In certain embodiments, the suicide spacer is a moiety having the structure:

[0181] Where Lp is an enzymatically cleavable bivalent peptide spacer, and A, D, L3 and R 2 As defined herein.

[0182] In a preferred embodiment, the suicide spacer is a moiety having the following structure:

[0183] Where Lp is an enzymatically cleavable bivalent peptide spacer, and D, L3 and R 2 As defined herein.

[0184] In other preferred embodiments, the suicide spacer is a moiety having the following structure:

[0185] Where Lp is an enzymatically cleavable bivalent peptide spacer, and D, L3 and R 2 As defined herein. In some embodiments, D is a quaternized tertiary amine-containing drug moiety, wherein the ammonium cation is optionally present as a zwitterion or with a monovalent anionic counterion.

[0186] As used herein, the term "hydrophilic moiety" refers to a moiety with hydrophilic properties that increases the water solubility of the drug moiety (D) when 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, C2-C6 alkyl and polysarcosine substituted with a group, such as having the formula Those wherein n is an integer between 2 and 25; and R is H, -CH3 or -CH2CH2C(=O)OH.

[0187] In some embodiments, the hydrophilic moiety comprises polyethylene glycol having the formula: Wherein 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 is H or optionally replaced by OH or C 1-4 Alkoxy-substituted C 1-4 alkyl, and m and n are each an integer between 2 and 25 (e.g., between 3 and 25). In some embodiments, the hydrophilic portion comprises

[0188] 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 complete immunoglobulins and can be derived from natural sources or from recombinant sources. Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed 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 is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the combination of immunoglobulins with 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). The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, a camelized antibody, or a chimeric antibody. These antibodies can have any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classification (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0189] The term "antibody fragment" or "antigen-binding fragment" or "functional fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an antigenic epitope (e.g., by 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), Camelid VHH domains, multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected, for example, by a synthetic linker (e.g., a short flexible polypeptide linker) and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, a scFv may have VL and VH variable regions in any order, eg, relative to the N-terminus and C-terminus of the polypeptide, and may comprise VL-linker-VH or may comprise VH-linker-VL.

[0190] 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 a number of well-known schemes, including those described by 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 a combination thereof, and Immunogenetics (IMGT) numbering (Lefranc, M.-P., The Immunogenetics of 1991). Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). In the combined Kabat and Jossian numbering scheme for a given CDR region (e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, or LC CDR3), in some embodiments, these CDRs correspond to amino acid residues defined as part of a Kabat CDR and amino acid residues defined as part of a Jossian CDR. As used herein, CDRs defined according to the "Jossian" numbering scheme are sometimes also referred to as "hypervariable loops."

[0191] 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 acid residues 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, the CDR comprises, for example, amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL or consists thereof. According to IMGT, the CDR amino acid residues in VH are numbered as approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered as approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (according to "Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0192] The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with a molecule. Epitope determinants typically consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate or sugar side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be "linear" or "conformational." The difference between conformational and linear epitopes is that binding to conformational epitopes (but not linear epitopes) is lost in the presence of denaturing solvents.

[0193] As used herein, the phrases "monoclonal antibody" or "monoclonal antibody composition" refer to polypeptides (including antibodies, bispecific antibodies, etc.) having substantially identical amino acid sequences or derived from the same genetic source. This term also includes preparations of antibody molecules having single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0194] As used herein, the phrase "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences, or mutant forms of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al. (2000. J Mol Biol [Molecular Biology Journal] 296, 57-86). The structure and position 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 (see, e.g., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), ed. Kabat et al.; Al Lazikani et al., (1997) J. Mol. Biol. 273:927-948); Kabat et al., (1991) ... Interest, 5th ed., 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)).

[0195] The human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or conservative substitutions to improve stability or production). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted into human framework sequences.

[0196] As used herein, the phrase "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) (the animals are transgenic or transchromosomal for human immunoglobulin genes) or hybridomas prepared therefrom; antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas); antibodies isolated from recombinant combinatorial human antibody libraries; and antibodies prepared, expressed, produced or isolated by any other means involving splicing all or part of human immunoglobulin genes, sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, but these sequences may not naturally occur in the human antibody germline library in vivo.

[0197] 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 domains of an antibody. Optionally, the Fc region may include a CH4 domain present in some antibody classes. The Fc region may include the entire hinge region of an antibody constant domain. In one embodiment, the present invention comprises the Fc region and CH1 region of an antibody. In one embodiment, the present invention comprises the Fc region and CH3 region of an antibody. In another embodiment, the present invention comprises the Fc region, CH1 region, and Cκ / λ region from an antibody constant domain. In one embodiment, the binding molecule of the present 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 present invention disclosed herein may comprise changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to the light chain constant region domain (CL). Example 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.

[0198] As used herein, the term "binding specificity" refers to the ability of a single antibody binding site to react with one antigenic determinant but not with a different antigenic determinant. The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. The binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody.

[0199] 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 regions of the antibody "arms" interact with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0200] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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 uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within an antibody can be replaced by other amino acid residues from the same side chain family, and the altered antibodies can be tested using the functional assays described herein.

[0201] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, such as two nucleic acid molecules (such as two DNA molecules or two RNA molecules) or between two polypeptide molecules. When a subunit position in the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching positions or homologous positions; for example, if half of the positions in the two sequences (for example, five positions in a polymer of ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are matched or homologous, then the two sequences are 90% homologous. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein a fragment of the amino acid sequence in the comparison window may contain additions or deletions (for example, gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions) to optimally align the two sequences. The percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0202] The sequence comparison between two sequences and the determination of percent identity can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com). The Blossum 62 matrix or the PAM250 matrix is ​​used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com). A particularly preferred set of parameters (and the parameters that should be used unless otherwise specified) is the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0203] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS [Computer Applications in Biosciences] 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0204] The nucleic acid sequences and protein sequences described herein can be used as "query sequences" to search public databases, 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. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0205] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of a compound of the present invention and at least one, and optionally more than one, other pharmaceutically acceptable chemical component (such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent, and / or excipient).

[0206] As used herein, the term "optical isomer" or "stereoisomer" refers to any of the various stereoisomeric conformations in which a given compound of the present invention may exist and includes geometric isomers. It should be understood that substituents may be attached to chiral centers of carbon atoms. The term "chiral" refers to a molecule that has the property of non-superimposability on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner. Therefore, the present invention includes enantiomers, diastereomers, or racemates of the compound. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of an enantiomeric pair is a "racemic" mixture. The term is used to refer to a racemic mixture where appropriate. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is specified according to the Cahn-Lngold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by R or S. Resolved compounds of unknown absolute conformation can be assigned (+) or (-) depending on the direction (dextrorotatory or levorotatory) in which they rotate plane-polarized light of the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)-.

[0207] 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 delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329). Except in the case where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0208] As used herein, the term "pharmaceutically acceptable salts" refers to salts that do not abrogate the biological activity and properties of the compounds of the present invention and do not cause significant irritation to a subject to which they are administered.

[0209] 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, cows, horses, sheep, goats, pigs; rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. Typically, the subject is a human.

[0210] The term "subject in need of such treatment" refers to a subject who would benefit biologically, medically, or in quality of life from such treatment.

[0211] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder, in one embodiment, refers to alleviating the disease or disorder (i.e., slowing, arresting, or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or reducing at least one physical parameter, including those that cannot be discerned by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both.

[0212] As used herein, the terms "prevent," "preventing," or "prevention" of any disease or disorder refer to prophylactic treatment of a disease or disorder; or delaying the onset or progression of a disease or disorder.

[0213] The terms "therapeutically effective amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to achieve a desired result (i.e., reduce or inhibit enzyme or protein activity, alleviate symptoms, relieve symptoms or conditions, delay disease progression, reduce tumor size, inhibit tumor growth, prevent metastasis, inhibit or prevent viral, bacterial, fungal or parasitic infection). In some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically effective amount induces or causes side effects, but only those that are acceptable to a healthcare provider in light of the patient's condition. A therapeutically effective amount can be determined by initially administering a low dose and then incrementally increasing the dose until the desired effect is achieved. A "prophylactically effective dose" or "prophylactically effective amount" of a molecule of the invention can prevent the onset of disease symptoms, including symptoms associated with cancer. A "therapeutically effective dose" or "therapeutically effective amount" of a molecule of the invention can result in a decrease in the severity of disease symptoms, including symptoms associated with cancer.

[0214] The compound names provided herein were obtained using ChemBioDraw Ultra (version 14.0).

[0215] As used herein, the terms "a," "an," "the," and similar terms used in the context of the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0216] Any formula given herein is also intended to represent non-labeled forms of compounds as well as isotopically labeled forms. Except that one or more atoms are replaced by atoms with selected atomic weights or mass numbers, isotopically labeled compounds have the structure described by the formula given herein. Isotopes that can be incorporated into the compounds of the present invention include, for example, isotopes of hydrogen.

[0217] Linker-drug group

[0218] The linker-drug group of the present invention is a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0219]

[0220] in:

[0221] R 1 It is a reactive group;

[0222] L1 is a bridging spacer;

[0223] Lp is a divalent peptide spacer;

[0224] G-L2-A is a suicide spacer;

[0225] R 2 It is the hydrophilic part;

[0226] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0227] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0228] L3 is the spacer portion;

[0229] and

[0230] 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.

[0231] Certain aspects and examples of the linker-drug groups of the present invention are provided in the following list of enumerated embodiments. It will be appreciated that the features specified in each embodiment can be combined with other specified features to provide further embodiments of the present invention.

[0232] Example 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0233] R 1 It is a reactive group;

[0234] L1 is a bridging spacer;

[0235] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0236] G-L2-A is a suicide spacer;

[0237] R 2 It is the hydrophilic part;

[0238] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0239] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0240] L3 is the spacer portion;

[0241] and

[0242] 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.

[0243] Example 2. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0244] R 1 It is a reactive group;

[0245] L1 is a bridging spacer;

[0246] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0247] described The group is selected from:

[0248] in The * indicates the point of attachment to the N or O of the drug moiety, The *** indicates the attachment point with Lp;

[0249] R 2 It is the hydrophilic part;

[0250] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0251] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0252] L3 is the spacer portion;

[0253] and

[0254] 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.

[0255] Example 3. Compounds of formula (I) having the structure of formula (II):

[0256]

[0257] or a pharmaceutically acceptable salt thereof,

[0258] in:

[0259] R 1 It is a reactive group;

[0260] L1 is a bridging spacer;

[0261] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0262] R 2 It is the hydrophilic part;

[0263] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0264] L3 is the spacer portion;

[0265] and

[0266] 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.

[0267] Embodiment 4. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 3, wherein:

[0268] 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,

[0269] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;*-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3C(=O)(CH2) m -**;*-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 X1(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(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 points;

[0270] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0271] Each R 3 Independently selected from H and C1-C6 alkyl;

[0272] R 4 is 2-pyridyl or 4-pyridyl;

[0273] Each R 5 Independently selected from H, C1-C6 alkyl, F, Cl, and -OH;

[0274] Each R 6 Independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2 and -OH;

[0275] Each R 7 Independently selected from H, C 1-6 Alkyl, fluorine, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C 1-4 Alkoxy and C substituted by -C(=O)OH 1-4 alkyl;

[0276] X1 is

[0277] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0278] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0279] 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;

[0280] Lp is a divalent peptide spacer comprising an amino acid residue selected from the group consisting of glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

[0281] 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 aare independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0282] L3 is a structure The spacer portion,

[0283] in

[0284] (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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0285] 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 point of attachment to R 2 the attachment point; or

[0286] (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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0287] 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 points;

[0288] and

[0289] The * of L3 indicates that the 2 attachment points;

[0290] and

[0291] 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.

[0292] Embodiment 5. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 4, wherein:

[0293] R 1 yes -ONH2,

[0294] 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 * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 attachment points;

[0295] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0296] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0297] 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;

[0298] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0299] L3 is a structure The spacer portion,

[0300] in

[0301] 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)NRb -**, -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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0302] 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 point of attachment to R 2 attachment points;

[0303] and

[0304] The * of L3 indicates that the 2 attachment points;

[0305] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0306] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0307] and

[0308] 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.

[0309] Embodiment 6. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 5, wherein:

[0310] R 1 yes

[0311] 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 * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 attachment points;

[0312] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0313] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0314] 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;

[0315] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0316] L3 is a structure The spacer portion,

[0317] in

[0318] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0319] 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 point of attachment to R 2 attachment points;

[0320] and

[0321] The * of L3 indicates that the 2 attachment points;

[0322] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0323] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0324] and

[0325] 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.

[0326] Embodiment 7. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 6, wherein:

[0327] R 1 yes

[0328] 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 * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 1 attachment points;

[0329] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0330] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0331] 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;

[0332] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0333] L3 is a structure The spacer portion,

[0334] in

[0335] 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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0336] 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 point of attachment to R 2 attachment points;

[0337] and

[0338] The * of L3 indicates that the 2 attachment points;

[0339] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0340] A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

[0341] and

[0342] 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.

[0343] Embodiment 8. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 7, wherein:

[0344] R 1 yes

[0345] 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 * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 1 attachment points;

[0346] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0347] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0348] 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;

[0349] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0350] L3 is a structure The spacer portion,

[0351] in

[0352] 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 and wherein the ** of said W indicates the point of attachment to X;

[0353] X is ***-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 points;

[0354] and

[0355] The * of L3 indicates that the 2 attachment points;

[0356] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0357] A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

[0358] and

[0359] 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.

[0360] Embodiment 9. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 8, wherein R 1 is a reactive group selected from Table 1 or Table 2.

[0361] Embodiment 10. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9,

[0362] in:

[0363] 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,

[0364] Embodiment 11. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9,

[0365] in:

[0366] 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,

[0367] Embodiment 12. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9,

[0368] in:

[0369] R 1 yes -ONH2,

[0370] Embodiment 13. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9,

[0371] in:

[0372] R 1 yes -ONH2,

[0373] Embodiment 14. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9, wherein R1 is

[0374] Embodiment 15. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9, wherein R 1 It is -ONH2.

[0375] Embodiment 16. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9, wherein: R 1 yes

[0376] Embodiment 17. The compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 9, wherein:

[0377] R 1 yes

[0378] Embodiment 18. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0379] or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0380] Embodiment 19. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0381] or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0382] Embodiment 20. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0383] or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0384] Embodiment 21. A compound of formula (I) according to any one of embodiments 1 to 8, having the structure:

[0385] or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0386] Embodiment 22. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0387] or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0388] Embodiment 23. A compound of formula (I) according to any one of embodiments 1 to 8, having the structure:

[0389] 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.

[0390] Embodiment 24. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0391] or a pharmaceutically acceptable salt thereof, wherein R is H, -CH3 or -CH2CH2C(=O)OH.

[0392] Embodiment 25. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0393] 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.

[0394] Embodiment 26. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0395] or a pharmaceutically acceptable salt thereof.

[0396] Embodiment 27. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0397] or a pharmaceutically acceptable salt thereof.

[0398] Embodiment 28. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0399] or a pharmaceutically acceptable salt thereof.

[0400] Embodiment 29. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0401] or a pharmaceutically acceptable salt thereof.

[0402] Embodiment 30. The compound of formula (I) according to any one of embodiments 1 to 8, which has the structure:

[0403] or a pharmaceutically acceptable salt thereof.

[0404] Embodiment 31. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of Embodiments 1 to 8, having the structure of a compound in any one of Tables 4A-4C included herein.

[0405] Example 32. A linker-drug group linker having formula (I), wherein the linker has a structure of formula (V),

[0406]

[0407] in

[0408] L1 is a bridging spacer;

[0409] Lp is a divalent peptide spacer;

[0410] G-L2-A is a suicide spacer;

[0411] R 2 It is the hydrophilic part;

[0412] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0413] 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 and wherein the ** of A indicates the point of attachment to L2,

[0414] and

[0415] L3 is the spacer portion.

[0416] Embodiment 33. The connector of embodiment 32, wherein:

[0417] L1 is a bridging spacer;

[0418] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0419] G-L2-A is a suicide spacer;

[0420] R 2 It is the hydrophilic part;

[0421] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0422] 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 and wherein the ** of A indicates the point of attachment to L2,

[0423] and

[0424] L3 is the spacer portion.

[0425] Embodiment 34. The connector of embodiment 32 or 33, wherein:

[0426] L1 is a bridging spacer;

[0427] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0428] described The group is selected from:

[0429] in The * indicates the point of attachment to the N or O of the drug moiety, The *** indicates the attachment point with Lp;

[0430] R 2 It is the hydrophilic part;

[0431] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0432] A is a bond, **-OC(=O)-, **-OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-or **-OC(=O)N(CH3)C(Ra )2C(R a )2N(CH3)C(=O)-, where each R a independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of A indicates the point of attachment to L2,

[0433] and

[0434] L3 is the spacer portion.

[0435] Embodiment 35. The connector of any one of embodiments 32 to 34, wherein:

[0436] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;*-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;*-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 X1(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) nNHC(=O)(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(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;

[0437] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0438] Each R 3 Independently selected from H and C1-C6 alkyl;

[0439] X1 is

[0440] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0441] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0442] 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;

[0443] Lp is a divalent peptide spacer comprising an amino acid residue selected from the group consisting of glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

[0444] 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 and wherein the ** of A indicates the point of attachment to L2;

[0445] L3 is a structure The spacer portion,

[0446] in

[0447] (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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0448] 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 point of attachment to R 2 the attachment point; or

[0449] (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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0450] 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 points;

[0451] and

[0452] The * of L3 indicates that the 2 attachment point.

[0453] Embodiment 36. The connector of any one of embodiments 32 to 35, wherein:

[0454] 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 * of L1 indicates the attachment point to Lp;

[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 selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0459] L3 is a structure The spacer portion,

[0460] in

[0461] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0462] 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 point of attachment to R 2 attachment points;

[0463] and

[0464] The * of L3 indicates that the 2 attachment points;

[0465] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0466] and

[0467] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of A indicates the point of attachment to L2.

[0468] Embodiment 37. The connector of any one of embodiments 32 to 36, wherein:

[0469] 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 * of L1 indicates the attachment point to Lp;

[0470] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0471] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0472] 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;

[0473] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0474] L3 is a structure The spacer portion,

[0475] in

[0476] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0477] 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 point of attachment to R 2 attachment points;

[0478] and

[0479] The * of L3 indicates that the 2 attachment points;

[0480] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0481] and

[0482] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of A indicates the point of attachment to L2.

[0483] Embodiment 38. The connector of any one of embodiments 32 to 37, wherein:

[0484] 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 * of L1 indicates the attachment point to Lp;

[0485] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0486] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0487] 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;

[0488] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0489] L3 is a structure The spacer portion,

[0490] in

[0491] 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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0492] 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 point of attachment to R 2 attachment points;

[0493] and

[0494] The * of L3 indicates that the 2 attachment points;

[0495] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0496] and

[0497] A is a bond or **-OC(=O)-, wherein the ** of A indicates the point of attachment to L2.

[0498] Embodiment 39. The connector of any one of embodiments 32 to 38, wherein:

[0499] 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 * of L1 indicates the attachment point to Lp;

[0500] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0501] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0502] 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;

[0503] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0504] L3 is a structure The spacer portion,

[0505] in

[0506] 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 and wherein the ** of said W indicates the point of attachment to X;

[0507] X is ***-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 points;

[0508] and

[0509] The * of L3 indicates that the 2 attachment points;

[0510] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or The hydrophilic portion of the group-substituted C2-C6 alkyl group;

[0511] and

[0512] A is a bond or **-OC(=O)-, wherein the ** of A indicates the point of attachment to L2.

[0513] Example 40. A linker of formula (V) having a structure of formula (VI)

[0514]

[0515] in

[0516] L1 is a bridging spacer;

[0517] Lp is a divalent peptide spacer;

[0518] R 2 It is the hydrophilic part;

[0519] 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,

[0520] and

[0521] L3 is the spacer portion.

[0522] Embodiment 41. The connector of embodiment 40, wherein:

[0523] L1 is a bridging spacer;

[0524] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0525] R 2 It is the hydrophilic part;

[0526] 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,

[0527] and

[0528] L3 is the spacer portion.

[0529] Embodiment 42. The connector of embodiment 40 or 41, wherein:

[0530] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;*-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;*-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 X1(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;*-C(=O)((CH2) m O)t (CH2) n NHC(=O)(CH2) n X1(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;

[0531] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, or a hydrophilic portion of polysarcosine;

[0532] Each R 3 Independently selected from H and C1-C6 alkyl;

[0533] X1 is

[0534] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0535] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0536] 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;

[0537] Lp is a divalent peptide spacer comprising an amino acid residue selected from the group consisting of glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

[0538] 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 aIndependently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl;

[0539] L3 is a structure The spacer portion,

[0540] in

[0541] (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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0542] 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 point of attachment to R 2 the attachment point; or

[0543] (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)NRb -**, -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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0544] 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 points;

[0545] and

[0546] The * of L3 indicates that the 2 attachment point.

[0547] Embodiment 43. The connector of any one of embodiments 40 to 42, wherein:

[0548] 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 * of L1 indicates the attachment point to Lp;

[0549] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0550] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0551] 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;

[0552] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group;

[0553] L3 is a structure The spacer portion,

[0554] in

[0555] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0556] 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 point of attachment to R2 attachment points;

[0557] and

[0558] The * of L3 indicates that the 2 attachment points;

[0559] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, or a hydrophilic portion of polysarcosine;

[0560] and

[0561] 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.

[0562] Embodiment 44. The connector of any one of embodiments 40 to 43, wherein:

[0563] 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 * of L1 indicates the attachment point to Lp;

[0564] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0565] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0566] 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;

[0567] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group;

[0568] L3 is a structure The spacer portion,

[0569] in

[0570] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0571] 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 point of attachment to R 2 attachment points;

[0572] and

[0573] The * of L3 indicates that the 2 attachment points;

[0574] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a hydrophilic portion of a group-substituted C2-C6 alkyl group or polysarcosine;

[0575] and

[0576] 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.

[0577] Embodiment 45. The connector of any one of embodiments 40 to 44, wherein:

[0578] 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 * of L1 indicates the attachment point to Lp;

[0579] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0580] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0581] 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;

[0582] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group;

[0583] L3 is a structure The spacer portion,

[0584] in

[0585] 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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0586] 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 point of attachment to R 2 attachment points;

[0587] and

[0588] The * of L3 indicates that the 2 attachment points;

[0589] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, or a hydrophilic portion of polysarcosine;

[0590] and

[0591] A is a bond or -OC(=O)-.

[0592] Embodiment 46. The connector of any one of embodiments 40 to 45, wherein:

[0593] 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 * of L1 indicates the attachment point to Lp;

[0594] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0595] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0596] 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;

[0597] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group;

[0598] L3 is a structure The spacer portion,

[0599] in

[0600] 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 and wherein the ** of said W indicates the point of attachment to X;

[0601] X is ***-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 points;

[0602] and

[0603] The * of L3 indicates that the 2 attachment points;

[0604] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, or a hydrophilic portion of polysarcosine;

[0605] and

[0606] A is a bond or -OC(=O)-.

[0607] Embodiment 47. The linker of any one of embodiments 32 to 46, having the structure:

[0608] in

[0609] R is H, -CH3 or -CH2CH2C(=O)OH.

[0610] Embodiment 48. The linker of any one of embodiments 32 to 46, having the structure:

[0611] in

[0612] R is H, -CH3 or -CH2CH2C(=O)OH.

[0613] Embodiment 49. The linker of any one of embodiments 32 to 46, having the structure:

[0614] in

[0615] R is H, -CH3 or -CH2CH2C(=O)OH.

[0616] Embodiment 50. The linker of any one of embodiments 32 to 46, having the structure:

[0617] wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0618] Embodiment 51. The linker of any one of embodiments 32 to 46, having the structure:

[0619] wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

[0620] Embodiment 52. The linker of any one of embodiments 32 to 46, having the structure:

[0621] in

[0622] Xa is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

[0623] Embodiment 53. The linker of any one of embodiments 32 to 46, having the structure:

[0624] in

[0625] R is H, -CH3 or -CH2CH2C(=O)OH.

[0626] Embodiment 54. The linker of any one of embodiments 32 to 46, having the structure:

[0627] in

[0628] Xb is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

[0629] Embodiment 55. The linker of any one of embodiments 32 to 46, having the structure:

[0630]

[0631] Embodiment 56. The linker of any one of embodiments 32 to 46, having the structure:

[0632]

[0633] Embodiment 57. The linker of any one of embodiments 32 to 46, having the structure:

[0634]

[0635] Embodiment 58. The linker of any one of embodiments 32 to 46, having the structure:

[0636]

[0637] Embodiment 59. The linker of any one of embodiments 32 to 46, having the structure:

[0638]

[0639] For the purpose of illustration, the general reaction scheme described herein provides a possible approach to 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 scheme and discussed below, other starting materials and reagents can be easily replaced to provide various derivatives and / or reaction conditions. In addition, according to this disclosure, conventional chemistry well known to those skilled in the art can be used to further modify many compounds prepared by the following methods.

[0640] By way of example, a general synthesis of compounds having formula (II) is shown in Scheme 1 below.

[0641] Solution 1

[0642]

[0643] Antibody drug conjugates of the present invention

[0644] The present invention provides antibody drug conjugates, also referred to herein as immunoconjugates, comprising a linker comprising one or more hydrophilic moieties.

[0645] The antibody drug conjugate of the present invention has the structure of formula (III):

[0646]

[0647]

[0648] in:

[0649] Ab is an antibody or its fragment;

[0650] R 100 is a coupling group;

[0651] L1 is a bridging spacer;

[0652] Lp is a divalent peptide spacer;

[0653] G-L2-A is a suicide spacer;

[0654] R 2 It is the hydrophilic part;

[0655] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0656] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0657] L3 is the spacer portion;

[0658] 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,

[0659] and

[0660] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0661] Certain aspects and examples of the antibody drug conjugates of the present invention are provided in the following list of enumerated embodiments. It will be appreciated that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.

[0662] Example 60. An immunoconjugate of formula (III), wherein:

[0663] Ab is an antibody or its fragment;

[0664] R 100 is a coupling group;

[0665] L1 is a bridging spacer;

[0666] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0667] G-L2-A is a suicide spacer;

[0668] R 2 It is the hydrophilic part;

[0669] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0670] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0671] L3 is the spacer portion;

[0672] 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,

[0673] and

[0674] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0675] Embodiment 61. The immunoconjugate of formula (III) as described in embodiment 60, wherein:

[0676] Ab is an antibody or its fragment;

[0677] R 100 is a coupling group;

[0678] L1 is a bridging spacer;

[0679] Lp is a bivalent peptide spacer containing one to four amino acid residues;

[0680] described The group is selected from:

[0681] in The * indicates the point of attachment to the N or O of the drug moiety, The *** indicates the attachment point with Lp;

[0682] R 2 It is the hydrophilic part;

[0683] L2 is a bond, methylene, neopentylene or C2-C3 alkenylene;

[0684] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0685] L3 is the spacer portion;

[0686] 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,

[0687] and

[0688] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0689] Embodiment 62. The immunoconjugate of any one of embodiments 60 to 61 having formula (III), which has a structure of formula (IV),

[0690]

[0691] in:

[0692] Ab is an antibody or its fragment;

[0693] R 100 is a coupling group;

[0694] L1 is a bridging spacer;

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

[0696] R 2 It is the hydrophilic part;

[0697] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0698] L3 is the spacer portion;

[0699] 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,

[0700] and

[0701] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0702] Embodiment 63. The immunoconjugate of formula (III) of any one of embodiments 60 to 62, wherein:

[0703] Ab is an antibody or its fragment;

[0704] 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 *** indicates the point of attachment to the Ab;

[0705] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n-**;*-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**;*-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 X1(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(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 100 attachment points;

[0706] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine;

[0707] Each R 3 Independently selected from H and C1-C6 alkyl;

[0708] R 4 is 2-pyridyl or 4-pyridyl;

[0709] Each R 5 Independently selected from H, C1-C6 alkyl, F, Cl, and -OH;

[0710] Each R 6 Independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2 and -OH;

[0711] Each R 7 Independently selected from H, C 1-6 Alkyl, fluorine, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C 1-4 Alkoxy and C substituted by -C(=O)OH 1-4 alkyl;

[0712] X1 is

[0713] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0714] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0715] 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;

[0716] Lp is a divalent peptide spacer comprising an amino acid residue selected from the group consisting of valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

[0717] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0718] L3 is a structure The spacer portion,

[0719] in

[0720] (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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0721] 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 point of attachment to R 2 the attachment point; or

[0722] (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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0723] 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 points;

[0724] and

[0725] The * of L3 indicates that the 2 attachment points;

[0726] 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,

[0727] and

[0728] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0729] Embodiment 64. The immunoconjugate of formula (III) of any one of embodiments 60 to 63, wherein:

[0730] Ab is an antibody or its fragment;

[0731] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab;

[0732] 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 * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 100 attachment points;

[0733] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0734] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0735] 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;

[0736] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0737] L3 is a structure The spacer portion,

[0738] in

[0739] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0740] 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 point of attachment to R 2 attachment points;

[0741] and

[0742] The * of L3 indicates that the 2 attachment points;

[0743] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine;

[0744] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0745] 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,

[0746] and

[0747] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0748] Embodiment 65. The immunoconjugate of formula (III) of any one of embodiments 60 to 64, wherein:

[0749] Ab is an antibody or its fragment;

[0750] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab;

[0751] 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 * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 100 attachment points;

[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 selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0756] L3 is a structure The spacer portion,

[0757] in

[0758] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0759] 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 point of attachment to R 2 attachment points;

[0760] and

[0761] The * of L3 indicates that the 2 attachment points;

[0762] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or a C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine;

[0763] 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D;

[0764] 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,

[0765] and

[0766] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0767] Embodiment 66. The immunoconjugate of formula (III) according to any one of embodiments 60 to 65, wherein:

[0768] Ab is an antibody or its fragment;

[0769] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab;

[0770] 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 * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 100 attachment points;

[0771] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

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

[0773] 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;

[0774] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0775] L3 is a structure The spacer portion,

[0776] in

[0777] 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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0778] 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 point of attachment to R 2 attachment points;

[0779] and

[0780] The * of L3 indicates that the 2 attachment points;

[0781] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine;

[0782] A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

[0783] 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,

[0784] and

[0785] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0786] Embodiment 67. The immunoconjugate of formula (III) according to any one of embodiments 60 to 66, wherein:

[0787] Ab is an antibody or its fragment;

[0788] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab;

[0789] 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 * of L1 indicates the attachment point with Lp and the ** of L1 indicates the attachment point with R 100 attachment points;

[0790] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

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

[0792] 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;

[0793] Lp is selected from A divalent peptide spacer of wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G;

[0794] L3 is a structure The spacer portion,

[0795] in

[0796] 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 and wherein the ** of said W indicates the point of attachment to X;

[0797] X is ***-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 points;

[0798] and

[0799] The * of L3 indicates that the 2 attachment points;

[0800] R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, a C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine;

[0801] A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

[0802] 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,

[0803] and

[0804] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0805] Embodiment 68. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 62, wherein R 100 It is a coupling group.

[0806] Embodiment 69. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 63, wherein

[0807] R 100 yes -S-, -C(=O)-, -ON=***, -NHC(=O)CH2-***, -S(=O)2CH2CH2-***, -(CH2)2S(=O)2CH2CH2-***, -NHS(=O)2CH2CH 2- ***、

[0808] -NHC(=O)CH2CH2-***, -CH2NHCH2CH2-***, -NHCH2CH2-***, where R 100 The *** indicates the point of attachment to the Ab.

[0809] Embodiment 70. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 63, wherein

[0810] 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 *** indicates the point of attachment to the Ab.

[0811] Embodiment 71. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 63, wherein

[0812] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab.

[0813] Embodiment 72. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 63, wherein

[0814] R 100 yes wherein R 100 The *** indicates the point of attachment to the Ab.

[0815] Embodiment 73. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0816] in

[0817] 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.

[0818] Embodiment 74. The immunoconjugate of formula (III) of any one of embodiments 60 to 72, having the structure:

[0819] in

[0820] 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.

[0821] Embodiment 75. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0822] wherein 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.

[0823] Embodiment 76. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0824]

[0825] in

[0826] 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.

[0827] Embodiment 77. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0828] in

[0829] 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.

[0830] Embodiment 78. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0831] wherein 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.

[0832] Embodiment 79. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0833] wherein 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.

[0834] Embodiment 80. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0835] in

[0836] 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.

[0837] Embodiment 81. The immunoconjugate of formula (III) of any one of embodiments 60 to 72, having the structure:

[0838] wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0839] Embodiment 82. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure: wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0840] Embodiment 83. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0841] wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0842] Embodiment 84. The immunoconjugate of formula (III) of any one of embodiments 60 to 72, having the structure:

[0843] wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0844] Embodiment 85. The immunoconjugate of any one of embodiments 60 to 72 having formula (III), which has the structure:

[0845] wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0846] 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 will be appreciated that the features specified in each embodiment can be combined with other specified features to provide additional embodiments of the present invention.

[0847] Embodiment 86. The compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 2, the linker of formula (V) as described in any one of embodiments 32 to 39, and the immunoconjugate of formula (III) as described in any one of embodiments 60 to 61, wherein:

[0848] G is Wherein the * of G indicates the attachment point with L2, the ** of G indicates the attachment point with L3, and the *** of G indicates the attachment point with Lp.

[0849] Embodiment 87. The compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 2, the linker of formula (V) as described in any one of embodiments 32 to 39, and the immunoconjugate of formula (III) as described in any one of embodiments 60 to 61, wherein:

[0850] G is Wherein the * of G indicates the attachment point with L2, the ** of G indicates the attachment point with L3, and the *** of G indicates the attachment point with Lp.

[0851] Embodiment 88. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein:

[0852] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n-**; *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**; *-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 X1(CH2) m -**; *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**; *-C(=O)(CH2) m NHC(=O)(CH2) n -**; *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**; *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**; *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(CH2) n -**;

[0853] *-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 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0854] Embodiment 89. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein:

[0855] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;*-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 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0856] Embodiment 90. The compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, the linker of formula (V) as described in any one of embodiments 32 to 46, and the immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein:

[0857] L1 is *-C(=O)(CH2) m O(CH2) m -**;*-C(=O)((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m -**;*-C(=O)NH((CH2) m O) t (CH2) n -**;*-C(=O)(CH2) m NH(CH2) m -**;*-C(=O)(CH2) m NH(CH2) n C(=O)-**; or *-C(=O)(CH2) m NHC(=O)(CH2) n -**, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0858] Embodiment 91. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein:

[0859] 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 * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1(if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0860] Embodiment 92. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein 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 attachment point with R 1 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0861] Embodiment 93. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein L1 is *-C(=O)((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 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0862] Embodiment 94. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein L1 is *-C(=O)(CH2) m -**, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0863] Embodiment 95. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72, wherein L1 is *-C(=O)NH((CH2) mO) t (CH2) n -**, where the * of L1 indicates the attachment point with Lp, and the ** of L1 indicates the attachment point with R 1 (if any) the attachment point or the **indicator of the L1 and R 100 Attachment point (if any).

[0864] Embodiment 96. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein Lp is a divalent peptide spacer, e.g., an enzymatically cleavable spacer.

[0865] Embodiment 97. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein Lp is a bivalent peptide spacer comprising amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0866] Embodiment 98. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein Lp is a bivalent peptide spacer comprising one to four amino acid residues (e.g., two to four amino acid residues).

[0867] Embodiment 99. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein Lp is a bivalent 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.

[0868] Embodiment 100. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0869] Lp is selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0870] Embodiment 101. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0871] Lp is wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0872] Embodiment 102. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0873] Lp is wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0874] Embodiment 103. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0875] Lp is wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0876] Embodiment 104. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0877] Lp is wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0878] Embodiment 105. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 95, wherein:

[0879] Lp is wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of formula (II) or the ** of Lp indicates the point of attachment to G of formula (I).

[0880] Embodiment 106. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 105, wherein L2 is a bond, methylene, neopentylene, or C2-C3 alkenylene.

[0881] Embodiment 107. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 105, wherein L2 is a bond or a methylene group.

[0882] Embodiment 108. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 105, wherein L2 is a bond.

[0883] Embodiment 109. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 105, wherein L2 is methylene.

[0884] Embodiment 110. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein:

[0885] 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.

[0886] Embodiment 111. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein A is a bond or -OC(=O).

[0887] Embodiment 112. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein A is a bond.

[0888] Embodiment 113. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein A is -OC(=O).

[0889] Embodiment 114. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein:

[0890] A is

[0891] Embodiment 115. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 109, wherein:

[0892] A is -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)- or

[0893] -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.

[0894] Embodiment 116. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein L3 is a spacer moiety.

[0895] Embodiment 117. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0896] L3 is a structure The spacer portion,

[0897] in

[0898] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0899] 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 point of attachment to R 2 attachment points;

[0900] and

[0901] The * of L3 indicates that the 2 attachment point.

[0902] Embodiment 118. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0903] L3 is a structure The spacer portion,

[0904] in

[0905] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0906] X is a bond;

[0907] and

[0908] The * of L3 indicates that the 2 attachment point.

[0909] Embodiment 119. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0910] L3 is a structure The spacer portion,

[0911] in

[0912] 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0913] X is triazole, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 attachment points;

[0914] and

[0915] The * of L3 indicates that the 2 attachment point.

[0916] Embodiment 120. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0917] L3 is a structure The spacer portion,

[0918] in

[0919] 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 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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said W indicates the point of attachment to X;

[0920] X is ***-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 points;

[0921] and

[0922] The * of L3 indicates that the 2 attachment point.

[0923] Embodiment 121. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0924] L3 is a structure The spacer portion,

[0925] in

[0926] 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 and wherein the ** of said W indicates the point of attachment to X;

[0927] 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 point of attachment to R 2 attachment points;

[0928] and

[0929] The * of L3 indicates that the 2 attachment point.

[0930] Embodiment 122. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0931] L3 is a structure The spacer portion,

[0932] in

[0933] 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 and wherein the ** of said W indicates the point of attachment to X;

[0934] X is a bond;

[0935] and

[0936] The * of L3 indicates that the 2 attachment point.

[0937] Embodiment 123. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0938] L3 is a structure The spacer portion,

[0939] in

[0940] 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 and wherein the ** of said W indicates the point of attachment to X;

[0941] X is triazole, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R 2 attachment points;

[0942] and

[0943] The * of L3 indicates that the 2 attachment point.

[0944] Embodiment 124. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 115, wherein:

[0945] L3 is a structure The spacer portion,

[0946] in

[0947] 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 and wherein the ** of said W indicates the point of attachment to X;

[0948] X is ***-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 points;

[0949] and

[0950] The * of L3 indicates that the 2 attachment point.

[0951] Embodiment 125. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It is the hydrophilic part.

[0952] Embodiment 126. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, A C2-C6 alkyl group substituted with a group, and a hydrophilic portion of polysarcosine.

[0953] Embodiment 127. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It's sugar.

[0954] Embodiment 128. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It is an oligosaccharide.

[0955] Embodiment 129. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It's a polypeptide.

[0956] Embodiment 130. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It is a polyalkylene glycol.

[0957] Embodiment 131. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2It 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.

[0958] Embodiment 132. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It has the structure -((CH2) m O) t R"-polyalkylene glycol, wherein R" is H, CH3 or CH2CH2C(=O)OH, m is 1-10 and t is 4-40.

[0959] Embodiment 133. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It's polyethylene glycol.

[0960] Embodiment 134. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein R 2 It has the structure -(OCH2CH2) t R' is a polyethylene glycol wherein R' is OH, OCH3 or OCH2CH2C(=O)OH and t is 4-40.

[0961] Embodiment 135. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 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.

[0962] Embodiment 136. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein:

[0963] R 2 yes (where n is an integer between 1 and 6), wherein R 2 The * or wavy line indicates the attachment point to the X or L3.

[0964] Embodiment 137. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein:

[0965] R 2 yes wherein R 2 The * indicates the attachment point to X or L3.

[0966] Embodiment 138. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein:

[0967] R 2 yes where R 2 The * indicates the attachment point to X or L3.

[0968] Embodiment 139. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 124, wherein:

[0969] R 2 yes where R 2 The * indicates the attachment point to X or L3.

[0970] Embodiment 140. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 139, wherein each R 3 Independently selected from H and C1-C6 alkyl.

[0971] Embodiment 141. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 139, wherein each R 3 It’s H.

[0972] Embodiment 142. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 139, wherein each R 3 Independently selected from C1-C6 alkyl.

[0973] Embodiment 143. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 142, wherein:

[0974] X1 is

[0975] Embodiment 144. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 142, wherein:

[0976] X1 is

[0977] Embodiment 145. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 144, wherein:

[0978] Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0979] Embodiment 146. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 144, wherein:

[0980] Each m is independently selected from 1, 2, 3, 4, and 5.

[0981] Embodiment 147. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 144, wherein:

[0982] Each m is independently selected from 1, 2, and 3.

[0983] Embodiment 148. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 147, wherein:

[0984] Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0985] Embodiment 149. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 147, wherein:

[0986] Each n is independently selected from 1, 2, 3, 4 and 5.

[0987] Embodiment 150. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 147, wherein:

[0988] Each n is independently selected from 1, 2, and 3.

[0989] Embodiment 151. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 150, wherein:

[0990] 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.

[0991] Embodiment 152. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 150, wherein:

[0992] 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.

[0993] Embodiment 153. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 150, wherein:

[0994] 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.

[0995] Embodiment 154. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, a linker of formula (V) as described in any one of embodiments 32 to 46, and an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 150, wherein:

[0996] 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.

[0997] Embodiment 155. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0998] Embodiment 156. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0999] Embodiment 157. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[1000] Embodiment 158. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[1001] Embodiment 159. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, 6, 7, or 8.

[1002] Embodiment 160. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, 4, 5, or 6.

[1003] Embodiment 161. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1, 2, 3, or 4.

[1004] Embodiment 162. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 1 or 2.

[1005] Embodiment 163. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 2.

[1006] Embodiment 164. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 4.

[1007] Embodiment 165. The immunoconjugate of formula (III) of any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 6.

[1008] Embodiment 166. The immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 154, wherein y is 8.

[1009] Embodiment 167. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a drug moiety.

[1010] Embodiment 168. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein

[1011] 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.

[1012] Embodiment 169. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety.

[1013] Embodiment 170. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein

[1014] D is a hydrophobic 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 (eg, when attached to A, D may be a quaternary ammonium).

[1015] Embodiment 171. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 7.

[1016] Embodiment 172. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 6.

[1017] Embodiment 173. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 5.

[1018] Embodiment 174. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 4.

[1019] Embodiment 175. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 3.

[1020] Embodiment 176. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 1.5 to 2.

[1021] Embodiment 177. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 2 to 7.

[1022] Embodiment 178. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 2 to 6.

[1023] Embodiment 179. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 2 to 5.

[1024] Embodiment 180. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 2 to 4.

[1025] Embodiment 181. A compound of formula (I) as described in any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is a hydrophobic drug moiety having an SlogP value of 2 to 3.

[1026] Embodiment 182. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is auristatin.

[1027] Embodiment 183. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is

[1028]

[1029] Embodiment 184. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is not an MCL-1 inhibitor.

[1030] Embodiment 185. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is not a BCL-2 inhibitor.

[1031] Embodiment 186. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or any one of embodiments 86 to 166, wherein D is not a BCL-XL inhibitor.

[1032] Embodiment 187. A compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 17, or an immunoconjugate of formula (III) as described in any one of embodiments 60 to 72 or embodiments 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 a linker selected from L2 to L208 disclosed herein (e.g., in Tables 4A-4C), such as

[1033] The wavy line indicates the point of attachment to D.

[1034] Embodiment 188. A linker-drug group having a structure of formula (I) as described in any one of embodiments 32 to 46, which is derived from any linker selected from L2 to L208 described herein, for example,

[1035]

[1036] Wherein the wavy line indicates the point of attachment to D and the * indicates the point of attachment to the antibody or fragment thereof.

[1037] Conjugation method

[1038] The present invention provides various methods for conjugating the linker-drug groups of the present invention to antibodies or antibody fragments to produce antibody drug conjugates comprising a linker having one or more hydrophilic moieties.

[1039] The general reaction scheme for forming an antibody drug conjugate having formula (III) is shown below in Scheme 2:

[1040] Option 2

[1041]

[1042] Where: RG2 is a reactive group that is compatible with R 1 The groups react to form the corresponding R 100Groups (such groups are shown in Table 1). D, R 1 , L1, Lp, Ab, y and R 100 As defined herein.

[1043] Scheme 3 further illustrates this general method for forming an antibody drug conjugate having formula (III), wherein the antibody comprises a 1 A reactive group (RG2) that reacts with a group (as defined herein) to react via R 100 A group (as defined herein) covalently attaches the linker-drug group to the antibody. For illustrative purposes only, Scheme 3 shows an antibody with four RG2 groups.

[1044] Option 3

[1045]

[1046] In one aspect, the linker-drug group is conjugated to the antibody via a modified cysteine ​​residue in the antibody (see, e.g., WO2014 / 124316). Scheme 4 illustrates such a method for forming an antibody drug conjugate having formula (III), wherein the free thiol group generated by the engineered cysteine ​​residue in the antibody is conjugated to R 1 Group (where R 1 is maleimide) to react via R 100 Group (where R 100 The linker-drug group is covalently attached to the antibody by a succinimide ring. For illustration purposes only, Scheme 4 shows an antibody with four free thiol groups.

[1047] Option 4

[1048]

[1049] In another aspect, the linker-drug group is conjugated to the antibody via a lysine residue in the antibody. Scheme 5 illustrates such a method for forming an antibody drug conjugate having formula (III), wherein the free amine group from the lysine residue in the antibody is conjugated to R 1 Group (where R 1 is NHS ester, pentafluorophenyl or tetrafluorophenyl) to react via R 100 Group (where R 100 The linker-drug group is covalently attached to the antibody (wherein the linker-drug group is an amide). For illustrative purposes only, Scheme 5 shows an antibody with four amine groups.

[1050] Option 5

[1051]

[1052] On the other hand, the linker-drug group is conjugated to the antibody via forming an oxime bridge at the naturally occurring disulfide bridge of the antibody. By first reducing the interchain disulfide bridges of the antibody and then bridging to form a keto bridge using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone), an oxime bridge is formed. Subsequently, the linker-drug group is reacted with a hydroxylamine to form an oxime bond (oxime bridge), which attaches the linker-drug group to the antibody (see, for example, WO2014 / 083505). Scheme 6 shows this method for forming an antibody drug conjugate with formula (III).

[1053] Option 6

[1054]

[1055] The general reaction scheme for forming the antibody drug conjugate having formula (IV) is shown below in Scheme 7:

[1056] Option 7

[1057]

[1058] Where: RG2 is a reactive group that is compatible with R 1 The groups react 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 herein.

[1059] Scheme 8 further illustrates this general method for forming an antibody drug conjugate having formula (IV), wherein the antibody comprises a 1 A reactive group (RG2) that reacts with a group (as defined herein) to react via R 100 A group (as defined herein) covalently attaches the linker-drug group to the antibody. For illustrative purposes only, Scheme 8 shows an antibody with four RG2 groups.

[1060] Option 8

[1061]

[1062] In one aspect, the linker-drug group is conjugated to the antibody via a modified cysteine ​​residue in the antibody (see, e.g., WO2014 / 124316). Scheme 9 illustrates such a method for forming an antibody drug conjugate having formula (IV), wherein the free thiol group generated by the engineered cysteine ​​residue in the antibody is conjugated to R 1Group (where R 1 is maleimide) to react via R 100 Group (where R 100 The linker-drug group is covalently attached to the antibody by a succinimide ring. For illustration purposes only, Scheme 9 shows an antibody with four free thiol groups.

[1063] Option 9

[1064]

[1065] In another aspect, the linker-drug group is conjugated to the antibody via a lysine residue in the antibody. Scheme 10 illustrates such a method for forming an antibody drug conjugate having formula (IV), wherein the free amine group from the lysine residue in the antibody is conjugated to R 1 Group (where R 1 is NHS ester, pentafluorophenyl or tetrafluorophenyl) to react via R 100 Group (where R 100 The linker-drug group is covalently attached to the antibody (wherein the linker-drug group is an amide). For illustrative purposes only, Scheme 10 shows an antibody with four amine groups.

[1066] Plan 10

[1067]

[1068] On the other hand, the linker-drug group is conjugated to the antibody via forming an oxime bridge at the naturally occurring disulfide bridge of the antibody. By first reducing the interchain disulfide bridges of the antibody and then bridging to form a keto bridge using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone), an oxime bridge is formed. Subsequently, the linker-drug group is reacted with a hydroxylamine to form an oxime bond (oxime bridge), which attaches the linker-drug group to the antibody (see, for example, WO2014 / 083505). Scheme 11 illustrates this method for forming an antibody drug conjugate with formula (IV).

[1069] Plan 11

[1070]

[1071] Also provided is the scheme of some aspects of the analytical methodology for evaluating antibody conjugates of the present invention.This analytical methodology and result can prove that conjugate has favourable character, for example, makes them easier to manufacture, easier to be applied to patient, more effective and / or potentially safer character to patient.An example is to determine molecular size by size exclusion chromatography (SEC), wherein the amount of desired antibody species in sample is relative to the amount of high molecular weight pollutants (for example, dimer, multimer or aggregation antibody) or low molecular weight pollutants (for example, antibody fragment, degradation product or independent antibody chain) present in sample is determined.Usually, expectation has higher amount of monomer and lower amount of for example aggregation antibody, this is due to the influence of for example aggregate on other properties of antibody sample, these other properties are such as but not limited to clearance rate, immunogenicity and toxicity.Further example is to determine hydrophobicity by hydrophobic interaction chromatography (HIC), wherein the hydrophobicity of sample is assessed relative to a group of standard antibodies of known properties. In general, it is desirable to have low hydrophobicity due to the effect of hydrophobicity on other properties of the antibody sample, such as, but not limited to, aggregation, aggregation over time, adhesion to surfaces, hepatotoxicity, clearance, and pharmacokinetic exposure. See Damle, NK, Nat Biotechnol. 2008; 26(8): 884-885; Singh, SK, Pharm Res. 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 the 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 having a hydrophobicity index of 0.8 or greater as determined by hydrophobic interaction chromatography are provided.

[1072] Antibody

[1073] The present invention provides antibody conjugates comprising antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to antigens (e.g., tumor antigens). Antibodies or antibody fragments (e.g., antigen binding fragments) of the present invention include, but are not limited to, human monoclonal antibodies or fragments thereof isolated as described in the Examples.

[1074] In certain embodiments, the present invention provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody or antibody fragment (e.g., antigen-binding fragment) comprising a VH domain having the amino acid sequence of SEQ ID NO: 7, 27, 47, 67, 87, 107, or 154. In certain embodiments, the present invention also provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody or antibody fragment (e.g., antigen-binding fragment) comprising a VH CDR having the amino acid sequence of any one of the VH CDRs listed in Table 3 below. In a specific embodiment, the present invention provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody comprising (or alternatively consisting of) one, two, three, four, five or more VH CDRs having the amino acid sequence of any one of the VH CDRs listed in Table 3 below.

[1075] The present invention provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody or antibody fragment (e.g., antigen-binding fragment) comprising a VL domain having the amino acid sequence of SEQ ID NO: 17, 37, 57, 77, 97, 117, or 166. The present invention also provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody or antibody fragment (e.g., antigen-binding fragment) comprising a VL CDR having the amino acid sequence of any one of the VL CDRs listed in Table 3 below. In particular, the present invention provides an antibody conjugate comprising an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin, the antibody or antibody fragment (e.g., antigen-binding fragment) comprising (or alternatively consisting of) one, two, three or more VL CDRs having the amino acid sequence of any one of the VL CDRs listed in Table 3 below.

[1076] Other antibodies or antibody fragments (e.g., antigen-binding fragments) of the invention comprise amino acids that have been mutated but have at least 60%, 70%, 80%, 90%, or 95% identity in the CDR regions to the CDR regions depicted in the sequences set forth in Table 3. In some embodiments, the antibodies comprise mutant amino acid sequences wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR regions have been mutated when compared to the CDR regions depicted in the sequences set forth in Table 3.

[1077] The present invention also provides antibody conjugates including antibodies or their antigen-binding fragments, wherein the antibodies or their antigen-binding fragments comprise modifications in the constant regions of heavy chains, light chains, or both heavy and light chains, wherein specific amino acid residues have been mutated into cysteine, also referred to herein as "CysMab" or "Cys" antibodies. As discussed above, the drug moiety can be conjugated to the cysteine ​​residues on the antibody in a site-specific manner and by controlling the quantity of the drug moiety ("DAR controlled"). For example, cysteine ​​modifications performed on antibodies for the purpose of site-specific control of immunoconjugation are disclosed in WO2014 / 124316, which is incorporated herein in its entirety.

[1078] In some embodiments, the antibodies have been modified at positions 152 and 375 of the heavy chain, wherein the positions are defined according to the EU numbering system. That is, the modifications are E152C and S375C. In other embodiments, the antibodies have been modified at position 360 of the heavy chain and at position 107 of the kappa light chain, wherein 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 human IgG1 heavy chain and kappa light chain constant regions in SEQ ID NOs: 148-150 of Table 3. Throughout Table 3, cysteine ​​modifications from the wild-type sequence are underlined.

[1079] The present invention also provides nucleic acid sequences encoding VH, VL, full-length heavy chains, and full-length light chains of antibodies that specifically bind to P-cadherin. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[1080] Table 3. Examples of anti-P-cadherin antibodies of the present invention

[1081]

[1082]

[1083]

[1084]

[1085]

[1086]

[1087]

[1088]

[1089]

[1090]

[1091]

[1092]

[1093]

[1094]

[1095]

[1096]

[1097]

[1098]

[1099]

[1100]

[1101]

[1102]

[1103]

[1104]

[1105]

[1106]

[1107]

[1108] Other antibodies of the invention include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but have at least 60%, 70%, 80%, 90% or 95% identity to the sequences set forth in Table 3. In some embodiments, 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the variable region depicted in the sequences set forth in Table 3, while retaining substantially the same therapeutic activity as the antibodies listed in Table 3.

[1109] In some embodiments, antibodies or antibody fragments (e.g., antigen binding fragments) useful in the immunoconjugates of the present invention include 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 [Natural Biotechnology] 2008, 26: 925-932). In one embodiment, the present invention provides modified antibodies or antibody fragments thereof comprising substitutions of one or more amino acids with cysteine ​​at positions described herein. The sites for cysteine ​​substitutions 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. The modified antibodies or fragments may have two or more cysteine ​​substitutions, and these substitutions may be used in combination with other antibody modifications and conjugation methods as described herein. Methods for inserting cysteine ​​at specific positions in 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 certain embodiments, the modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine ​​in its constant region selected from the group consisting of 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 of the heavy chain of the antibody or antibody fragment, and wherein the positions are numbered according to the EU system. In some embodiments, the modified antibodies or antibody fragments thereof comprise substitutions of one or more amino acids with cysteine ​​on their constant region selected from the group consisting of 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 said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain. In certain embodiments, the modified antibodies or antibody fragments thereof comprise substitutions of two or more amino acids with cysteine ​​on their constant region, wherein the combination comprises substitutions at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, or position 107 of the antibody light chain, and wherein said positions are numbered according to the EU system.In certain embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​in its constant region, wherein the substitution is at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, position 107 of the antibody light chain, position 165 of the antibody light chain, or position 159 of the antibody light chain, and wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain. In specific embodiments, 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 position 375 of the antibody heavy chain and position 152 of the antibody heavy chain, and wherein the positions are numbered according to the EU system. In specific embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​at position 360 of the antibody heavy chain, and wherein the positions are numbered according to the EU system. In other specific 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 wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain. Exemplary embodiments of these positions are shown in the constant region sequences disclosed in SEQ ID NOs: 148, 149, and 150. Specific examples of these positions are disclosed for the anti-P-cadherin antibody sequences in SEQ ID NOs: 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147.

[1110] Since 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 the 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 specific VH / VL pairing should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a specific full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a specific VH / VL pairing should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a specific full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence. Thus, in one aspect, the invention provides an antibody conjugate comprising an isolated monoclonal antibody, or an antigen-binding region thereof, having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 17, 37, 57, 77, 97, and 117; wherein the antibody specifically binds P-cadherin.

[1111] In another aspect, the invention provides an antibody conjugate 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 NOs: 9, 29, 49, 69, 89, and 109, which has been 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 NOs: 19, 39, 59, 79, 99, and 119, which has been optimized for expression in mammalian cells; or (ii) a functional protein comprising an antigen-binding portion thereof.

[1112] In another aspect, the present invention provides an antibody conjugate comprising a P-cadherin-binding antibody comprising heavy and light chain CDR1s, CDR2s, and CDR3s, or a combination thereof, as described in Table 3. The amino acid sequences of the VH CDR1s of the antibodies are set forth in SEQ ID NOs: 1, 21, 41, 61, 81, and 101. The amino acid sequences of the VH CDR2s of the antibodies are set forth in SEQ ID NOs: 2, 22, 42, 62, 82, and 102. The amino acid sequences of the VH CDR3s of the antibodies are set forth in SEQ ID NOs: 3, 23, 43, 63, 83, and 103. The amino acid sequences of the VL CDR1s of the antibodies are set forth in SEQ ID NOs: 11, 31, 51, 71, 91, and 111. The amino acid sequences of the VL CDR2s of the antibodies are set forth in SEQ ID NOs: 12, 32, 52, 72, 92, and 112. The amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 13, 33, 53, 73, 93, and 113.

[1113] Given that each of these antibodies can bind to P-cadherin and that antigen binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, CDR2, and CDR3 sequences and the 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 particular 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 particular VL sequence should be replaced with one or more structurally similar CDR sequences. It will be readily apparent to those of ordinary skill that novel VH and VL sequences can be generated by substituting one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences set forth herein for the monoclonal antibodies of the invention.

[1114] Thus, the present invention provides an isolated monoclonal antibody or antigen-binding region thereof, comprising: a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 21, 41, 61, 81, and 101; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 62, 82, and 102; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, and 103; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 31, 51, 71, 91, and 111; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, and 112; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[1115] In specific embodiments, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to P-cadherin comprises a heavy chain CDR1 of SEQ ID NO: 1; a heavy chain CDR2 of SEQ ID NO: 2; a heavy chain CDR3 of SEQ ID NO: 3; a light chain CDR1 of SEQ ID NO: 11; a light chain CDR2 of SEQ ID NO: 12; and a light chain CDR3 of SEQ ID NO: 13.

[1116] In another specific embodiment, an 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.

[1117] In yet another embodiment, an 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.

[1118] In additional embodiments, an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to P-cadherin comprises a heavy chain CDR1 of SEQ ID NO:61; a heavy chain CDR2 of SEQ ID NO:62; a heavy chain CDR3 of SEQ ID NO:63; a light chain CDR1 of SEQ ID NO:71; a light chain CDR2 of SEQ ID NO:72; and a light chain CDR3 of SEQ ID NO:73.

[1119] In another specific embodiment, an 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.

[1120] 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.

[1121] In certain embodiments, the antibody that specifically binds to P-cadherin is an antibody or antibody fragment (eg, antigen-binding fragment) described in Table 3.

[1122] 2. Further changes to the Fc region framework

[1123] The immunoconjugates of the present invention may comprise modified antibodies or antigen-binding fragments thereof, which further comprise modifications to framework residues within 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 approach is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are 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 from which the antibody was derived. In order to restore the framework region sequence to its germline conformation, the somatic mutation can be "reverted" to the germline sequence, for example, by site-directed mutagenesis. Such "reverted" antibodies are also intended to be encompassed by the present invention.

[1124] Another type of framework modification involves mutating one or more residues within the framework region or even within 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 by Carr et al.

[1125] In addition to modifications made in the framework or CDR regions or in alternatives to modifications made in the framework or CDR regions, the antibodies of the present invention can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular toxicity. In addition, 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 its glycosylation, thereby again altering one or more functional properties of the antibody. Each of these embodiments is described in more detail below.

[1126] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the CH1 hinge region is altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease antibody stability.

[1127] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is further described in U.S. Patent No. 6,165,745 to Ward et al.

[1128] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[1129] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[1130] In another embodiment, one or more amino acid residues are changed to change the ability of the antibody to fix complement. This method is described in, for example, PCT publication WO 94 / 29351 by Bodmer et al. In a specific embodiment, one or more amino acids of an antibody of the present invention or its antigen-binding fragment are replaced by one or more heterotypic amino acid residues. Heterotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of IgG1, IgG2, and IgG3 subclasses and the constant region of the light chain of the kappa isotype, as described in Jefferis et al., MAbs. [monoclonal antibodies] 1: 332-338 (2009).

[1131] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This approach is described, for example, in PCT Publication WO 00 / 42072 by Presta. In addition, the binding sites for FcγR1, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[1132] In yet another embodiment, the glycosylation of the antibody is modified. For example, an antibody without glycosylation can be prepared (i.e., the antibody lacks glycosylation). Glycosylation can be changed to, for example, increase the affinity of the antibody for the "antigen". Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which results in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[1133] Additionally or alternatively, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express 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 functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. Presta described in PCT publication WO 03 / 035835 a variant CHO cell line, Lec13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002), J. Biol. Chem. 277:26733-26740). Umana et al. described in PCT publication WO 99 / 54342 a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[1134] In another embodiment, the antibody is modified to increase its biological half-life. Various methods can be used. 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 biological half-life, the antibody can be modified within the CH1 or CL region to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[1135] 3. Antibody Production

[1136] Antibodies and antibody fragments thereof (e.g., antigen binding fragments) can be produced 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, by hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[1137] The present disclosure further provides polynucleotides encoding antibodies described herein, for example, polynucleotides encoding heavy or light chain variable regions or segments comprising 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 to a polynucleotide selected from the group consisting of SEQ ID NOs: 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 to a polynucleotide selected from the group consisting of SEQ ID NOs: 18, 38, 58, 78, 98, 118, and 153.

[1138] 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 to 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 to the polynucleotide of SEQ ID NO: 20, 40, 60, 80, 100, 120, or 154.

[1139] The polynucleotides of the present invention may encode only the variable region sequences of an antibody. They may 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. Other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of the heavy and light chains of an antibody, respectively.

[1140] The polynucleotide sequence can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding an antibody or binding fragment thereof (e.g., a sequence as 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. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introduction of mutations into a polynucleotide sequence by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (ed.), Freeman Press, New York, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (eds.), 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.

[1141] The present invention also provides expression vectors and host cells for producing the antibodies described herein. A variety of expression vectors can be used to express polynucleotides encoding antibody chains or binding fragments. Both viral-based vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, additional vectors (typically with expression cassettes for expressing proteins or RNA), and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Natural Genetics] 15:345, 1997). For example, non-viral vectors that can be used to express anti-P-cadherin polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3, 1, 4-D-Hydroxy-1, 1, 6 ... 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 retrovirus-, adenovirus-, adeno-associated virus-, herpes virus-based vectors, SV40-, papillomavirus-, HBP-, Epstein-Barr virus-, vaccinia virus-, and Semliki Forest virus (SFV)-based vectors. See, Brent et al., supra; Smith, Annu. Rev. Microbiol. [Annual Review of Microbiology] 49:807, 1995; and Rosenfeld et al., Cell [Cell] 68:143, 1992.

[1142] The selection of expression vectors depends on the expected host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably connected to the polynucleotide encoding the anti-P-cadherin antibody chain or fragment. In certain embodiments, an inducible promoter is used to prevent the inserted sequence from being expressed under conditions other than induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. The culture of the transformed organism can be expanded under non-inducing conditions without favoring a colony of coding sequences that better tolerate the expression product of the host cell. In addition to the promoter, other regulatory elements may also be needed or desired to efficiently express the antibody chain or fragment. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be increased by including an enhancer appropriate for the cell system being used (see, e.g., 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 the CMV enhancer can be used to increase expression in mammalian host cells.

[1143] The expression vector can also provide a secretion signal sequence site to form a fusion protein with the polypeptide encoded by the inserted antibody sequence. More commonly, the inserted antibody sequence is linked to the signal sequence before being included in the vector. The vector used to receive the sequences encoding the anti-P-cadherin antibody light chain and heavy chain variable domains sometimes also encodes the constant region or a portion thereof. Such vectors allow the variable region to be expressed as a fusion protein with the constant region, thereby resulting in the production of a complete antibody or fragment thereof. Typically, such constant regions are human.

[1144] The host cell for carrying and expressing the antibody chain can be prokaryotic or eukaryotic. Escherichia coli (E.coli) is a prokaryotic host that can be used for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include bacillus (such as Bacillus subtilis) and other Enterobacteriaceae (such as Salmonella, Serratia) and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (for example, replication origin) compatible with the host cell. In addition, there will be any number of multiple well-known promoters, such as lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system or promoter system from bacteriophage lambda. Promoter typically optionally uses operator sequence control expression, and has ribosome binding site sequence etc., to start and complete transcription and translation. Other microorganisms such as yeast can also be used for expressing antibody polypeptides of the present invention. Insect cells in combination with baculovirus vectors can also be used.

[1145] In some preferred embodiments, mammalian host cells are used to express and produce antibody polypeptides of the present invention. For example, they can be hybridoma cell lines (for example, myeloma hybridoma clones as described in the examples) expressing endogenous immunoglobulin genes or mammalian cell lines (for example, the SP2 / 0 myeloma cells exemplified below) carrying exogenous expression vectors. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. Mammalian tissue cell culture expression of polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones [from genes to clones], VCH Publishers [VCH publishers], New York, NY, 1987. Expression vectors for mammalian host cells may contain expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. [Immunol. Review] 89: 49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or regulatable or adjustable. 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 (e.g., human CMV immediate early promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[1146] The method for introducing the expression vector containing the polynucleotide sequence of interest varies according to the type of cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts (generally referring to Sambrook et al., 2012, MOLECULAR CLONING:A LABORATORY MANUAL [molecular cloning: laboratory manual], 1-4 volumes, Cold Spring Harbor Press [Cold Spring Harbor Experiment Press], New York). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated conversion, injection and microinjection, bombardment, virion, immunoliposome, polycationic acid conjugates, naked DNA, artificial virion, with the fusion of herpes virus structural protein VP22 (Elliot and O'Hare, Cell [cell] 88:223,1997), the DNA uptake and in vitro transduction of medicament enhancement. For the long-term high yield production of recombinant protein, stable expression is generally desired. For example, expression vectors of the present invention that contain viral replication origin or endogenous expression element and selective marker gene can be used to prepare the cell line of stably expressing antibody chain or binding fragment.After introducing carrier, cell can be made to grow 1-2 days in enrichment medium, be converted to selective medium afterwards.The purpose of selective marker is to give selection resistance, and its existence allows the growth of the cell of the sequence introduced in selective medium of successful expression.Can use the tissue culture technique that is suitable for cell type to breed resistance, stable transfection cell.

[1147] Therapeutic uses and treatments

[1148] The antibody conjugates provided can be used in a variety of applications, including but not limited to the treatment of cancer. In certain embodiments, the antibody conjugates provided herein can be used to inhibit tumor growth, reduce tumor volume, induce differentiation, and / or reduce the tumorigenicity of tumors. The methods of use can be in vitro, ex vivo, or in vivo methods.

[1149] In some embodiments, provided herein are methods for treating, preventing, or alleviating a disease (e.g., cancer) in a subject (e.g., a human patient) in need thereof by administering any of the antibody conjugates described herein to the subject. Also provided are uses of the antibody conjugates of the present invention for treating or preventing a disease in a subject (e.g., a human patient). Additionally provided are uses of the antibody conjugates for treating or preventing a disease in a subject. In some embodiments, provided are antibody conjugates for the preparation of a medicament for treating or preventing a disease in a subject. In certain embodiments, the disease treated with the antibody conjugates is cancer.

[1150] In one aspect, the immunoconjugates described herein can be used to treat solid tumors. Examples of solid tumors include malignant tumors of various organ systems, for example, sarcomas, adenocarcinomas, blastomas, and cancers, such as those affecting the liver, lungs, breasts, lymph nodes, gallbladder (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, small cell lung cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. In one embodiment, the cancer is a melanoma, for example, an advanced melanoma. Examples of other cancers that can be treated include: bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, anal cancer, peritoneal cancer, 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 cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), pediatric solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, neuroendocrine tumors (including carcinoid tumors, gastrinomas, and pancreatic islet cell carcinomas), mesothelioma, schwannoma (including acoustic neuroma), meningioma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including cancers induced by asbestos), and combinations of said cancers.

[1151] In another aspect, the immunoconjugates described herein can be used to treat hematological cancers. Hematological cancers include leukemias, lymphomas, and malignant lymphoproliferative disorders that affect the blood, bone marrow, and lymphatic system.

[1152] Leukemia can be classified as acute leukemia and chronic leukemia.Acute leukemia can be further classified as acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).Other related conditions include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), which is a diversified set of hematological disorders combined by the invalid production (or dysplasia) of bone marrow blood cells and the risk of being converted into AML.

[1153] Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

[1154] In some embodiments, the cancer is a hematological cancer, including, but not limited to, e.g., acute leukemias, including, but not limited to, e.g., B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia ("ALL"); one or more chronic leukemias, including, but not limited to, e.g., chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematological cancers or hematological conditions, including, but not limited to, e.g., B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell leukemia, The present invention also includes but is not limited to leukemia, myelodysplasia, myelodysplasia, and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and a collection of various blood conditions that are associated with the ineffective production (or developmental abnormalities) of myeloid blood cells. Additional diseases associated with tumor antigen expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases that express tumor antigens as described herein. The methods and compositions of the present invention can also be used to treat or prevent metastatic lesions of the above-mentioned cancers.

[1155] In certain embodiments, the cancer is characterized by cells expressing a target tumor antigen, and the antibody or antibody fragment (e.g., antigen binding fragment) of the antibody conjugate binds to the target tumor antigen. In some embodiments, the immunoconjugates 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, detecting RNA expression levels of tumor antigens, and the like.

[1156] 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 alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testosterone or PRSS21); Endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); folate receptor alpha; neural cell adhesion molecule (NCAM); prostatic enzyme; prostatic acid phosphatase (PAP); mutated elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100);Oncogene fusion protein composed of the breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl 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 beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXO RF61); anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycosylceramide (GloboH); mammary differentiation antigen (NY-BR-1); adrenergic receptor beta 3 (ADRB3); pan-connexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor 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-related antigen 1; tumor protein p53 (p53); p53 mutants; prostate-specific protein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galactosylprotein 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTE RT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomas viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2);Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (mut hsp70-2); leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A, member 2 (LILRA2); CD300 molecule-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 with an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); and immunoglobulin lambda-like polypeptide 1 (IGLL1); CD184; LGR5; AXL; RON; CD35 2 / SLAMf6; KAAG-1; 5T4; c-Met; ITGA3; endosialin; CD166; SAIL (c15orf54); NaPi2b; DLL3; CD133; FZD7; antiadhesin; PD-L1; SLITRK6; nectin-4; FGFR2; FGFR3; FGFR4; CEACAM1; CEACAM5; CD74; STEAP-1; PMEL17; Muc16; FcRH5; TENB2; Ly6E; ETBR; 158P1D7; 161P2F10B; 191p4d12; 162p1e6; Notch3; PTK7; and EFNA4.

[1157] Tumor-supporting antigens

[1158] In some embodiments, an immunoconjugate as described herein may comprise an antigen binding domain (eg, an antibody or antibody fragment) that binds a tumor-supporting antigen (eg, a tumor-supporting antigen as described herein).

[1159] In some embodiments, the tumor-supporting antigen is an antigen present in stromal cells, antigen-presenting cells, or myeloid-derived suppressor cells (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. In some embodiments, the stromal cell antigens are selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In an embodiment, MDSC antigens are 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 activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[1160] It is also contemplated that the antibody conjugates described herein can be used to treat 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, renal 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 occlusive pulmonary disease, or edema following burns, trauma, radiation, stroke, hypoxia or ischemia, diabetic nephropathy, Paget's disease, disease), photoaging (e.g., caused by ultraviolet radiation exposure of human skin), benign prostatic hypertrophy, certain microbial infections including microbial pathogens selected from the group consisting of adenovirus, hantavirus, Borrelia burgdorferi, Yersinia spp., and Bordetella pertussis, thrombosis caused by platelet aggregation, reproductive disorders (e.g., endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menorrhagia), acute and chronic kidney disease (including proliferative glomerulonephritis), hypertrophic scarring, endotoxic shock and fungal infections, familial adenomatous polyposis, myelodysplastic syndrome, aplastic anemia, ischemic injury, fibrosis of the lung, kidney, or liver, infantile hypertrophic pyloric stenosis, urinary tract obstructive syndrome, psoriatic arthritis.

[1161] Methods of administration of such antibody conjugates include, but are not limited to, parenteral (e.g., intravenous) administration, e.g., bolus injection or continuous infusion over a period of time, oral administration, intramuscular administration, intratumoral administration, intramuscular administration, intraperitoneal administration, intracerebrospinal administration, subcutaneous administration, intraarticular administration, intrasynovial administration, lymph node injection, or intrathecal administration.

[1162] For the treatment of disease, the appropriate dosage of the antibody conjugates 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, previous therapy, the patient's clinical history, etc. The antibody conjugates can be administered once or over a series of treatments, lasting from a few days to several months, or until a cure is achieved or a reduction in the disease state is achieved (e.g., a decrease in tumor size). The optimal dosing regimen can be calculated from the cumulative measurement of the drug in the patient and will vary depending on the relative efficacy of the specific antibody conjugate. In certain 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 can be administered once or multiple times daily, weekly, monthly, or annually. In certain embodiments, the antibody conjugates of the present invention are administered once every two weeks or once every three weeks. In certain embodiments, the antibody conjugates of the present invention are only administered once. The treating physician can estimate repeat dosing rates based on measured residence times and concentrations of the drug in body fluids or tissues.

[1163] Pharmaceutical composition

[1164] To prepare pharmaceutical or sterile compositions comprising one or more antibody conjugates described herein, the provided antibody conjugates may be mixed with a pharmaceutically acceptable carrier or excipient.

[1165] 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, e.g., 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 Dekke, New York, 1993; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekke, 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).

[1166] In some embodiments, the pharmaceutical composition comprising the antibody conjugate of the present invention is a lyophilized formulation. In certain 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 certain 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 certain 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 injection, for example, with water or saline. In a specific embodiment, the solution comprises the antibody conjugate, histidine, sucrose, and polysorbate 20 at a pH of about 5.0. In another specific embodiment, the solution comprises the antibody conjugate, sodium succinate, and polysorbate 20. In another specific embodiment, the solution comprises the antibody conjugate, dehydrated trehalose, citrate dehydrate, citric acid, and polysorbate 8 at a pH of about 6.6. For intravenous administration, the resulting solution is generally further diluted in a carrier solution.

[1167] 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 biomatrix. In certain embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Therefore, the amount of biologic delivered depends in part on the specific entity and the severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, England, 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 Engl. J. Med. 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).

[1168] The appropriate dosage is determined by the clinician, for example, using parameters or factors known in the art or suspected of affecting treatment or expected to affect treatment. Typically, the dosage starts with an amount slightly less than the optimal dose and is increased in small increments thereafter until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic measurements include those of symptoms (e.g., inflammation) or the levels of inflammatory cytokines produced.

[1169] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention being employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors known in the medical arts.

[1170] Compositions comprising the antibody conjugates of the invention can be provided by continuous infusion, or by dosages spaced apart for example, one day, one week, 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. Dosages can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. A specific dosage regimen is one that involves a maximum dose or frequency of administration that avoids significant undesirable side effects.

[1171] For the antibody conjugates of the present invention, the dosage administered to the patient can be 0.0001 mg / kg to 100 mg / kg patient body weight. The dosage can be between 0.001 mg / kg patient body weight and 50 mg / kg patient body weight, between 0.005 mg / kg patient body weight and 20 mg / kg patient body weight, between 0.01 mg / kg patient body weight and 20 mg / kg patient body weight, between 0.02 mg / kg patient body weight and 10 mg / kg patient body weight, between 0.05 mg / kg patient body weight and 5 mg / kg patient body weight, between 0.1 mg / kg patient body weight and 10 mg / kg patient body weight, between 0.1 mg / kg patient body weight and 8 mg / kg patient body weight, between 0.1 mg / kg patient body weight and 5 mg / kg patient body weight, between 0.1 mg / kg patient body weight and 2 mg / kg patient body weight, between 0.1 mg / kg patient body weight and 1 mg / kg patient body weight. The dosage of the antibody conjugate can be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg / kg.

[1172] Doses of the antibody conjugates of the invention can be repeated and each administration can be separated by 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. In some embodiments, the antibody conjugates of the invention are administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or less frequently. In specific embodiments, the dose of the immunoconjugates of the invention is repeated every 2 weeks.

[1173] The effective amount for a particular patient can vary depending on factors such as the condition being treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects (see, e.g., Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, England, 2001).

[1174] The route of administration can be by, for example, topical or cutaneous application, injection or infusion by subcutaneous, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional administration, or by sustained release systems or implants (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; L (e.g., Rage, 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; U.S. Pat. Nos. 6,350,466 and 6,316,024). If desired, the composition may also include a solubilizing agent or a local anesthetic such as lidocaine to relieve pain at the injection site, or both. In addition, pulmonary administration may be employed, for example, by use of an inhaler or nebulizer, and formulations containing a nebulizing agent. See, e.g., 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 Publication Nos. 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.

[1175] Examples of such additional ingredients are well known in the art.

[1176] Methods of co-administration or treatment with a second therapeutic agent (e.g., a cytokine, a steroid, a chemotherapeutic agent, an antibiotic, or radiation) are known in the art (see, e.g., Hardman et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania). An effective amount of the therapeutic agent can reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40%, or at least 50%.

[1177] The additional therapy (e.g., a prophylactic or therapeutic agent) that can be administered in combination with the antibody conjugates of the invention can be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 hour apart, about 2 hours apart, about 3 hours apart, about 3 hours apart, about 4 hours apart, about 4 hours apart, about 5 hours apart, about 5 hours apart, about 6 hours apart, about 7 hours apart, about 8 hours apart, about 9 hours apart, or about 10 minutes apart. The two or more therapies can be administered at about the same patient visit, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. Two or more therapies can be administered during the same patient visit.

[1178] In certain embodiments, the antibody conjugates of the present invention can be formulated to ensure proper in vivo distribution. Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988), Biochem. Biophys. Res. Commun. [Biochemistry and Biophysics Research Communications] 153: 1038); antibodies (Bloeman et al., (1995) FEBS Lett. [European Biochemical Society Letters] 357: 140); Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123); JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[1179] The present invention provides a scheme for administering a pharmaceutical composition comprising the antibody conjugates of the present invention to a subject in need thereof, alone or in combination with other therapies. The therapy (e.g., a prophylactic or therapeutic agent) of the combination therapy of the present invention can be administered to the subject simultaneously or sequentially. The therapy (e.g., a prophylactic or therapeutic agent) of the combination therapy of the present invention can also be administered in cycles. Cycling 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.

[1180] The therapies (eg, prophylactic or therapeutic agents) of the combination therapies of the invention can be administered concurrently to a subject.

[1181] The term "concurrently" is not limited to administering therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means administering a pharmaceutical composition comprising an antibody of the present invention or a fragment thereof to a subject in such an order and within such a time interval that the antibody or antibody conjugate of the present invention can function together with one or more other therapies to provide an increased benefit compared to if they were administered separately. For example, each therapy can be administered to the subject at the same time or sequentially in any order at different time points; however, if not administered at the same time, they should be administered sufficiently close in time to provide the desired therapeutic or preventive effect. Each therapy can be administered to the subject separately in any appropriate form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 5 minutes apart, less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 hour apart, about 2 hours apart to about 3 hours apart, about 3 hours apart to about 4 hours apart, about 4 hours apart to about 5 hours apart, about 5 hours apart to about 6 hours apart, about 6 hours apart to about 7 hours apart, about 7 hours apart to about 8 hours apart, about 8 hours apart to about 9 hours apart, about 9 hours apart to about 10 hours apart, about 10 hours apart to about 11 hours apart, about 11 hours apart to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered in the same patient visit.

[1182] The prophylactic or therapeutic agents of the combination therapy can be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapy can be administered to the subject in parallel in separate pharmaceutical compositions. The prophylactic or therapeutic agents can be administered to the subject by the same or different routes of administration.

[1183] Examples

[1184] The present invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[1185] Temperatures are given in degrees Celsius. If not otherwise mentioned, all evaporations are carried out under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (=20-133 mbar). The structures of final products, intermediates, and starting materials are confirmed by standard analytical methods (e.g., microanalysis and spectroscopic characterization (e.g., MS, IR, NMR)). The abbreviations used are conventional in the art.

[1186] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of the present invention are commercially available or can be produced by organic synthesis methods known to those of ordinary skill in the art or can be produced by organic synthesis methods as described herein.

[1187] abbreviation:

[1188] The abbreviations used are those conventional in the art or the following:

[1189]

[1190]

[1191] Analytical methods

[1192] LC / MS data were acquired using the following instrument parameters:

[1193]

[1194] The method used to generate the LC / MS data is as follows:

[1195] Method A: 2min acid method

[1196] Eluent A1: 0.1% formic acid in water

[1197] Eluent B1: 0.1% formic acid in acetonitrile

[1198] Flow rate: 1.0mL / min

[1199] Stop time: 3.00min

[1200] pH: 2.6

[1201] Column: AcQuity UPLC BEH C18 1.7μm 2.1×50mm

[1202] Column temperature: 50°C

[1203] TAC: 210-400nm

[1204] Mass range: 120-1500Da

[1205] Scan time: 0.3s

[1206] gradient:

[1207] time %A(eluent A1) %B (eluent B1) 0.00 95 5 0.20 95 5 2.00 5 95 2.50 5 95 2.60 95 5 3.00 95 5

[1208] Method B: 2min alkaline method

[1209] Eluent A2: 5 mM ammonium hydroxide in water

[1210] Eluent B2: 5 mM ammonium hydroxide in acetonitrile

[1211] Flow rate: 1.0mL / min

[1212] Stop time: 3.00min

[1213] pH: 10.2

[1214] Column: AcQuity UPLC BEH C18 1.7μm 2.1×50mm

[1215] Column temperature: 50°C

[1216] TAC: 210-400nm

[1217] Mass range: 120-1500Da

[1218] Scan time: 0.3s

[1219] gradient:

[1220] time %A(eluent A2) %B (eluent B2) 0.00 95 5 0.20 95 5 2.00 5 95 2.50 5 95 2.60 95 5 3.00 95 5

[1221] Method C: 5min acid method

[1222] Flow rate: 1.0mL / min

[1223] Stop time: 5.20min

[1224] pH: 2.6

[1225] Column: AcQuity UPLC BEH C18 1.7μm 2.1×50mm

[1226] Column temperature: 50°C

[1227] TAC: 210-400nm

[1228] Mass range: 120-1500Da

[1229] Scan time: 0.3s

[1230] gradient:

[1231] time %A(eluent A1) %B (eluent B1) 0.00 98 2 4.40 2 98 5.15 2 98 5.19 98 2

[1232] HRMS data were acquired using an instrument with the following parameters:

[1233]

[1234] The methods used to generate HRMS data for linkers / payloads and synthetic intermediates are as follows:

[1235] Method D: 5min acid method

[1236] Flow rate: 1.0mL / min

[1237] Stop time: 5.2 minutes

[1238] pH: 2.6

[1239] Column: AcQuity UPLC BEH C18 1.7μm 2.1×50mm

[1240] Column temperature: 50°C

[1241] TAC: 210-400nm

[1242] Mass range: 300-4000Da

[1243] Treatment Range: n / a

[1244] Scan time: 0.5s

[1245] gradient:

[1246] time %A(eluent A2) %B (eluent B2) 0.00 98 2 4.40 2 98 5.15 2 98 5.19 98 2

[1247] The method used to generate HRMS data for the antibody-drug conjugate is as follows:

[1248] Method E: Protein Method

[1249] Flow rate: 1.0mL / min

[1250] Stop time: 3.30min

[1251] pH: 2.6

[1252] Column: ProSwift RP-3U 4.6x50 mm SS

[1253] Column temperature: 50°C

[1254] TAC: 210-400nm

[1255] Mass range: 600-3900Da

[1256] Processing range: 14000-170000Da

[1257] Scan time: 1.5s

[1258] gradient:

[1259] time %A(eluent A1) %B (eluent B1) 0.00 98 2 0.70 98 2 2.00 2 98 2.10 2 98 2.30 98 2 3.30 98 2

[1260] The following instrument parameters and a run time of 12 minutes were used to obtain Size exclusion chromatography data:

[1261]

[1262] Example 1: Synthesis of linker intermediates

[1263] 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-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (LI-1)

[1264]

[1265] Step 1: 2-(Bromomethyl)-4-nitrobenzoic acid Synthesis

[1266] At room temperature to 2-methyl-4-nitrobenzoic acid (300g, 1.5371mol) in CCl4 (3000mL) stirring solution add NBS (300.93mg, 1.6908mol) and AIBN (37.86mg, 0.2305mmol).Reaction mixture is stirred at 80 ℃ for 16h.Analyze and monitor reaction mixture by TLC.Reaction mixture is diluted with saturated NaHCO3 solution (2lit) and extracted with ethyl acetate (2x 2lit).The organic layer merged is dried over anhydrous sodium sulfate and under reduced pressure concentrated.Use the petroleum ether solution of 2%-3% ethyl acetate as eluent, by crude compound purification by silica gel column chromatography, and obtain 2-(bromomethyl)-4-nitrobenzoic acid (250g, 59% productive rate). 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).

[1267] Step 2: 4-Nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid Synthesis

[1268] To a mixture of 2-(bromomethyl)-4-nitrobenzoic acid (250 g, 0.9122 mol) in ACN (5000 mL) was added 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) at room temperature. The resulting mixture was heated to 80 ° C for 16 h. The reaction mixture was filtered through a celite pad and washed with ethyl acetate (2 lit). The filtrate was concentrated under reduced pressure. Saturated NaHCO3 solution (1 lit) was added to the crude compound obtained, 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)

[1269] Step 3: Methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate Synthesis

[1270] To a stirred solution of 4-nitro-2-((prop-2-alkynes-1-yloxy)methyl)benzoic acid (130g, 0.5527mol) in MeOH (1300mL) at 0°C, SOCl (526.08g, 320.78mL, 4.4219mol, d=1.64g / mL) was slowly added. The reaction was stirred at 70°C for 4h. The reaction solvent was evaporated under reduced pressure. The residue obtained was dissolved in ethyl acetate (1000mL) and washed with saturated NaHCO (600mL), water (500mL) and saline solution (500mL). The organic layer separated was dried over sodium sulfate, filtered and evaporated under reduced pressure to obtain methyl 4-nitro-2-((prop-2-alkynes-1-yloxy)methyl)benzoate (110g, 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).

[1271] Step 4: Methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate Synthesis

[1272] At room temperature, to a solution of methyl 4-nitro-2-((prop-2-alkynes-1-yloxy)methyl)benzoate (110 g, 0.4414 mol) in a mixture of EtOH (1100 mL) and H o (550 mL), Fe powder (197.21 g, 3.5310 mol) and NH cl (188.88 g, 3.5310 mol) were added. 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 half the volume. Ethyl acetate (1.5 lit) was added to the residue and the two layers were separated, and the water layer was extracted with ethyl acetate (2 lit). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. Purification by SiO2 column chromatography (15%-20% ethyl acetate in petroleum ether) gave methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate (70 g, 72% yield). 1 HNMR (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).

[1273] Step 5: (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol Synthesis

[1274] To a stirred solution of THF (1000 mL) was slowly added LiAlH4 (1 M in THF) (21.23 g, 798.2 mmol, 798.2 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 was stirred at room temperature for 4 h. The reaction mixture was cooled to 0 ° C., 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 resulting crude compound was purified by SiO2 column chromatography (35%-40% ethyl acetate in petroleum ether as eluent) to afford (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).

[1275] Step 6: Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate

[1276]

[1277] To a solution of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (1.92 g, 10.04 mmol, 1.0 equiv), (9H-fluoren-9-yl)methyl (S)-(1-amino-1-oxo-5-ureidopentan-2-yl)carbamate (3.99 g, 10.04 mmol, 1.0 equiv), 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 equiv) in DMF (10 mL) was added N , N-diisopropylethylamine (2.62 mL, 15.06 mmol, 1.5 equiv). After stirring at ambient temperature for 1 hour, the mixture was poured into water (200 mL). The resulting solid was filtered, rinsed with water, and dried under vacuum to obtain (9H-fluoren-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 acidic method - Method A).

[1278] Step 7: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide

[1279]

[1280] To (9H-fluoren-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 equiv) was added dimethylamine (2M in THF, 21.31 mL, 42.62 mmol, 4 equiv). After stirring at ambient temperature for 1.5 hours, the supernatant was decanted from the formed gummy residue. The residue was triturated with diethyl ether (3×50 mL) and the resulting solid was filtered, washed with diethyl ether, and dried in vacuo. (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 = 0.42 min (2 min acidic method - Method A).

[1281] Step 8: Synthesis of 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)

[1282]

[1283] 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 equiv), (tert-butoxycarbonyl)-L-valine (2.62 g, 12.05 mmol, 1.2 equiv), 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 equiv) in DMF (10 mL) was added N,N-diisopropylethylamine (3.50 mL, 20.08 mmol, 2.0 equiv). After stirring at ambient temperature for 2 h, the mixture was poured into water (200 mL), and the resulting suspension was washed with EtOAc (3 x 100 mL). The 1H- 1- hydroxy-2- methyl-1- oxo-3- methyl-2- oxo-4- thiophene-2- thiocyanate (LI-1) was purified by ISCO SiO2 chromatography (0-20% methanol / dichloromethane) to afford 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%). 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,ary l),6.76(d,J=8.9Hz,1H),5.95(t,J=5.8Hz,1H),5.38(s,2H),5.01(t,J=5.5Hz,1H),4.54(s,2H),4.45(dd,J=25.2,5.3Hz , 3H), 4.20 (d, J = 2.4 Hz, 2H), 3.83 (dd, J = 8.9, 6.7 Hz, 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).

[1284] Example 1-2: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (LI-2)

[1285]

[1286] Step 1: Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid

[1287]

[1288] To a solution of 6-nitroisobenzofuran-1(3H)-one (90 g, 502.43 mmol, 1.00 equiv) in MeOH (1000 mL) was added KOH (28.19 g, 502.43 mmol, 1.00 equiv) in HO (150 mL). The brown mixture was stirred at 25 ° C for 1.5 h. The brown mixture was concentrated under reduced pressure to give a residue and dissolved in DCM (2000 mL). TBDPSCl (296.91 g, 1.08 mol, 277.49 mL, 2.15 equiv) and imidazole (171.03 g, 2.51 mol, 5.00 equiv) were added to the mixture and stirred at 25 ° C for 12 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 1 / 1) and 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (34 g, 74.16 mmol, 14.76% yield) was obtained 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)

[1289] Step 2: Synthesis of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol

[1290]

[1291] To a mixture of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (41 g, 94.14 mmol, 1 eq) in THF (205 mL) was added BH 3 .THF (1 M, 470.68 mL, 5 eq). The yellow mixture was stirred at 60° C. for 2 h. MeOH (400 mL) was added to the mixture and concentrated under reduced pressure to give a residue, followed by addition of HO (200 mL) and DCM (300 mL), extraction with DCM (3×200 mL), washing with brine (300 mL), drying over anhydrous MgSO 4 , filtering, and concentrating under reduced pressure to give a residue. The residue was purified 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.

[1292] 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).

[1293] Step 3: Synthesis of 2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde

[1294]

[1295] To a solution of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol (34 g, 80.65 mmol, 1 eq) in DCM (450 mL) was added MnO (56.09 g, 645.22 mmol, 8 eq). The black mixture was stirred at 25° C. for 36 h. MeOH (400 mL) was added to the mixture and concentrated under reduced pressure to give a residue, followed by addition of H O (200 mL) and DCM (300 mL), extraction with DCM (3×200 mL), washing with brine (300 mL), drying over anhydrous MgSO , filtering, and concentration under reduced pressure to give a residue. The residue was purified by silica gel chromatography (CHCl=100%). 2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (30 g, 71.51 mmol, 88.7% yield) was obtained as a white solid.

[1296] 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)

[1297] Step 4: Synthesis of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine

[1298]

[1299] To a solution of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (12.6 g, 30.03 mmol, 1 eq) in DCM (130 mL) was added prop-2-yn-1-amine (4.14 g, 75.08 mmol, 4.81 mL, 2.5 eq) and MgSO (36.15 g, 300.33 mmol, 10 eq) and the suspension mixture was stirred at 25 ° C for 24 h. A small amount of the reaction solution was taken and treated with NaBH, and TLC showed that a new spot had formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-1-imine (12 g, crude product) 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).

[1300] (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-1-imine (12 g, 26.28 mmol, 1 eq) was dissolved in MeOH (100 mL) and THF (50 mL), followed by the addition of NaBH (1.49 g, 39.42 mmol, 1.5 eq) and the yellow mixture was stirred at -20 ° C for 2 h. LCMS showed that the desired compound was detected. The reaction mixture was quenched by adding 200 mL of MeOH at -20 ° C, then concentrated under reduced pressure to give a residue. The residue was dissolved in EtOAc 500 mL, washed with brine 150 mL, dried over anhydrous Na SO, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (eluent: 0%-10% ethyl acetate / petroleum ether gradient). N-(2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 18.45 mmol, 70% yield) was obtained as a light 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).

[1301] Step 5: Synthesis of (9H-fluoren-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate

[1302]

[1303] To a solution of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 19.62 mmol, 1 eq) and Fmoc-OSU (7.28 g, 21.59 mmol, 1.1 eq) in dioxane (90 mL) was added saturated NaHCO (90 mL) and the white suspension was stirred at 20° C. for 12 h. The reaction mixture was diluted with HO 150 mL and extracted twice with EtOAc (150 mL each). The combined organic layers were washed with brine 200 mL, dried over anhydrous NaSO, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (eluent: 0%-30% ethyl acetate / petroleum ether). (9H-Fluoren-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 as a white solid.

[1304] 1H NMR (400 MHz, chloroform-d) δ ppm 1.12 (s, 9H) 2.17 (br d, J=14.31 Hz, 1H) 3.87-4.97 (m, 9H) 6.98-8.28 (m, 21H).

[1305] Step 6: Synthesis of (9H-fluoren-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1306]

[1307] To an ice-bath cooled solution of (9H-fluoren-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate (5.0 g, 7.34 mmol, 1.0 equiv) in 10% AcOH / CHCl (100 mL) was added Zn (7.20 g, 110 mmol, 15 equiv). The ice bath was removed and the resulting mixture was stirred for 2 h, at which time it was filtered through a pad of Celite. The volatiles were removed in vacuo and the residue was dissolved in EtOAc, washed with NaHCO (sat), NaCl (sat), dried over MgSO, filtered, concentrated and (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.99 g, 62%) was obtained after ISCO SiO chromatography (0%-75% EtOAc / heptane). LCMS: MH = 651.6; R = 3.77 min (5 min acidic - Method C).

[1308] Step 7: Synthesis of (9H-fluoren-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1309]

[1310] To (9H-fluoren-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 equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanic acid (1.72 g, 4.59 mmol, 1.0 equiv) in CHCl (40 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.27 g, 9.18 mmol, 2.0 equiv). After stirring for 10 min, MeOH (1 mL) was added to make the solution homogeneous. The reaction was stirred for 16 h, the volatiles were removed in vacuo, and after purification by ISCO SiO chromatography (0%-15% MeOH / CH2Cl2), (9H-fluoren-9-yl)methyl (5-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.78 g, 60%) was obtained. LCMS: MH+ = 1008.8; Rt = 3.77 min (5 min acidic method - Method C).

[1311] Step 8: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

[1312]

[1313] To (9H-fluoren-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (1.60 g, 1.588 mmol, 1.0 equiv) was added 2M dimethylamine in MeOH (30 mL, 60 mmol, 37 equiv) and THF (10 mL). After standing for 3 h, the volatiles were removed in vacuo and the residue was triturated with EtO to remove the Fmoc deprotection byproduct. To the resulting solid was added CHCl (16 mL) and pyridine (4 mL), and to the heterogeneous solution was added propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equiv). After stirring for 30 min, additional propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equiv) was added. After stirring for an additional 20 minutes, MeOH (1 mL) was added to quench the remaining chloroformate and the volatiles were removed in vacuo. Purification by ISCO SiO chromatography (0%-15% MeOH / CH Cl) gave prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butyloxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (984 mg, 71%). LCMS: MH + = 867.8; Rt = 3.40 min (5 min acidic method - Method C). Step 9: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (LI-2)

[1314]

[1315] To a solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (984 mg, 1.135 mmol, 1.0 equiv) in THF (7.5 mL) was added 1.0 M tetrabutylammonium fluoride in THF (2.27 mL, 2.27 mmol, 2.0 equiv). After standing for 6 h, the volatiles were removed in vacuo and the residue was purified by ISCO SiO chromatography (0%-40% MeOH / CH2Cl2) to afford prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (629 mg, 88%). LCMS: MH+ = 629.6; Rt = 1.74 min (5 min acidic method - Method C).

[1316] Example 1-3: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3)

[1317]

[1318] Step 1: Synthesis of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide

[1319]

[1320] To a stirred suspension of 6-nitroisobenzofuran-1(3H)-one (500 g, 2.79 mol) in MeOH (1500 mL) was added MeNH2 (3.00 kg, 29.94 mol, 600 mL, 31.0% purity) 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 a residue. The product 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, crude) 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).

[1321] Step 2: Synthesis of (2-((methylamino)methyl)-4-nitrophenyl)methanol

[1322]

[1323] A solution of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560g, 2.66mol) in THF (5000mL) was cooled to 0°C, and BH3-Me2S (506g, 6.66mol) (2.0M in THF) was added dropwise for 60min, and the mixture was heated to 70°C for 5h. LCMS shows that the starting material is consumed. After completion, 4M HCl (1200mL) in methanol was added to the reaction mixture at 0°C, and heated at 65°C for 8h. The reaction mixture was cooled to 0°C, the solid was filtered and concentrated under reduced pressure. (2-((methylamino)methyl)-4-nitrophenyl)methanol (520g) was obtained as a white solid. LCMS: RT=0.742min, 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)

[1324] Step 3: Synthesis of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine

[1325]

[1326] A solution of (2-((methylamino)methyl)-4-nitrophenyl)methanol (520g, 2.65mol) and imidazole (721g, 10.6mol) in DCM (2600mL) was cooled to 0°C, then TBDPS-Cl (1.09kg, 3.98mol, 1.02L) was added dropwise, and the mixture was stirred for 2h. The mixture was poured into ice-cold water (1000mL) and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated under vacuum to give a crude product. The crude product was purified by silica gel chromatography (eluted with ethyl acetate: petroleum ether (from 10 / 1 to 1)) to give a residue. 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (600g) was obtained as a yellow liquid. LCMS: Product: RT = 0.910 min, MS m / z = 435.2 [M+H]+

[1327] 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)

[1328] Step 4: Synthesis of (9H-fluoren-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate

[1329]

[1330] To a solution of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (400 g, 920.3 mmol) in THF (4000 mL) was added Fmoc-OSU (341.5 g, 1.01 mol) and EtN (186.2 g, 1.84 mol, 256.2 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 NaSO, 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-fluoren-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]+.

[1331] 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).

[1332] Step 5: Synthesis of (9H-fluoren-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1333]

[1334] A solution of (9H-fluoren-9-yl)methyl(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (3.0 g, 4.57 mmol, 1.0 equiv) in MeOH (90 mL) and EtOAc (30 mL) was degassed and purged to a balloon of N2 via a three-way stopcock. After repeating the degassing / N2 purge twice, 10% Pd / C deGussa type (0.486 g, 0.457 mmol, 0.1 equiv) was added. The resulting mixture was degassed and purged to a balloon of 2H2 via a three-way stopcock. After repeating the degassing / H2 purge twice, the reaction was stirred under a balloon pressure of H2 for 4 hours. The reaction was degassed and purged to N2, filtered through a pad of celite, and further eluted with MeOH. After removal of the volatiles in vacuo and pumping under high vacuum, (9H-fluoren-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).

[1335] Step 6: Synthesis of (9H-fluoren-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1336]

[1337] To (9H-fluoren-9-yl)methyl(5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.86 g, 4.56 mmol, 1.0 equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanic acid (1.71 g, 4.56 mmol, 1.0 equiv) in 2:1 CHCl / MeOH (60 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.256 g, 9.12 mmol, 2.0 equiv). The homogeneous solution was stirred for 16 hours, at which time additional (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.95 g, 65%) was obtained after stirring for an additional 5 hours. LCMS: MH+ = 984.1; Rt = 1.54 min (2 min acidic method - Method A).

[1338] Step 7: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[1339]

[1340] To (9H-fluoren-9-yl)methyl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.05 g, 2.085 mmol, 1.0 equiv) in THF (10 mL) was added 2.0 M dimethylamine in MeOH (10.42 mL, 20.85 mmol, 10 equiv). After stirring for 16 h, the volatiles were removed in vacuo. The residue was dissolved in CHCl (20 mL) and DIEA (0.533 mL, 4.17 mmol, 2 equiv) and propargyl chloroformate (0.264 mL, 2.71 mmol, 1.3 equiv) were added. After stirring at room temperature for 16 h, the reaction was diluted with CH2Cl2 (20 mL), washed with NaHCO3 (sat), NaCl (sat), dried over MgSO4, filtered, concentrated, and purified by ISCO SiO2 chromatography (0%-15% MeOH / CH2Cl2) to give prop-2-yn-1-yl (5-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.04 g, 59%). LCMS: MH+ = 843.8; Rt = 1.35 min (2 min acidic method - Method A). Step 8: Synthesis of prop-2-yn-1-yl(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3)

[1341]

[1342] To a 0 °C solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.6 g, 1.90 mmol, 1.0 equiv) in THF (10.0 mL) was added 1.0 M tetrabutylammonium fluoride in THF (3.80 mL, 3.80 mmol, 2.0 equiv). After warming to room temperature and stirring for 16 h, the volatiles were removed in vacuo and the residue was dissolved in EtOAc, washed with NaHCO (sat), NaCl (sat), dried over MgSO, filtered, concentrated, and the residue was purified by ISCO SiO chromatography (0%-30% MeOH / CHCl) to give prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (LI-3) (1.0 g, 87%). LCMS: MH = 605.7; R = 0.81 min (2 min acidic - Method A).

[1343] Example 1-4: Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-(((prop-2-yn-1-yloxy)carbonyl)amino)acetamido)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (LI-4)

[1344]

[1345] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate

[1346]

[1347] To a solution of (4-amino-2-nitrophenyl)methanol (10 g, 59.5 mmol, 1.0 equiv), (9H-fluoren-9-yl)methyl (S)-(1-amino-1-oxo-5-ureidopentan-2-yl)carbamate (23.64 g, 59.5 mmol, 1.0 equiv), and 1-hydroxy-7-azabenzotriazole (8.50 g, 62.4 mmol, 1.05 equiv) in DMF (50 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (11.97 g, 62.4 mmol, 1.05 equiv). 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, rinsed with water, and dried under vacuum. (9H-Fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (31.49 g, 57.5 mmol, 97%) was obtained. LCMS: MH+ = 548; Rt = 2.02 min (5 min acidic method - Method C).

[1348] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide

[1349]

[1350] To a solution of (9H-fluoren-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 equiv) in DMF (50 mL) was added dimethylamine (2M in MeOH, 331 mL, 661 mmol, 11.5 equiv). After stirring at ambient temperature for 24 h, the volatiles were removed under vacuum and the residue was triturated with diethyl ether (3 x 2 L). The residue was dried under vacuum to give (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).

[1351] 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

[1352]

[1353] To a solution of (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide (10.89 g, 28.8 mmol, 1.0 equiv), (tert-butoxycarbonyl)-L-valine (6.25 g, 28.8 mmol, 1.0 equiv), and 1-hydroxy-7-azabenzotriazole (3.92 g, 28.8 mmol, 1.0 equiv) in DMF (40 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5.52 g, 28.8 mmol, 1.0 equiv). After stirring at ambient temperature for 24 hours, the mixture was added dropwise to water (2 L), stirred for 30 minutes, 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).

[1354] 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

[1355]

[1356] 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 equiv) and imidazole (15.52 g, 228 mmol, 10 equiv) in DMF (31 mL) was added tert-butyldimethylsilyl chloride (13.68 g, 90.76 mmol, 4.0 equiv). The resulting mixture was stirred at ambient temperature for 48 hours and then heated at 45 ° C for 4 hours. The mixture was poured into water and stirred for 96 hours. The solid was filtered and washed with water (2×100 mL) and dried under vacuum. 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 after purification by SiO2 ISCO chromatography (0%-30% methanol / dichloromethane). LCMS: MH+ = 639.6; Rt = 1.22 min (2 min acidic method - Method A).

[1357] 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

[1358]

[1359] Under nitrogen atmosphere, to tert-butyl ((S) -1- (((S) -1- ((4- (((tert-butyldimethylsilyl) oxy) methyl) -3- nitrophenyl) amino) -1- oxo -5- urea groups -2- bases) amino) -3- methyl -1- oxo butan- 2- bases) carbamate (8.02g, 12.56mmol, 1.0 equivalents) in methanol (250mL) solution, palladium on carbon (10wt%, 2.00g, 1.884mmol, 0.15 equivalents) is added. The mixture is placed under 1atm dihydrogen and stirred at ambient temperature for 18 hours. The mixture is filtered through celite and concentrated in vacuo. 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 after purification by SiO2 ISCO chromatography (0%-40% methanol / dichloromethane). LCMS: MH+ = 609.6; Rt = 2.65 min (5 min acidic method - Method C).

[1360] Step 6: Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-(((prop-2-yn-1-yloxy)carbonyl)amino)acetamido)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (LI-4)

[1361]

[1362] Step 6a):

[1363] To a solution of glycine (3.19g, 42.5mmol, 1.0 equivalent) in 2M aqueous sodium hydroxide solution (63.3mL, 127mmolNaOH, 3.0 equivalents) was added propargyl chloroformate (5.0g, 42.5mmol, 1.0 equivalent). The resulting mixture was stirred at ambient temperature for 3 hours. The mixture was extracted with ethyl acetate (3x 250mL). The combined organic layer was dried over magnesium sulfate, filtered, and volatiles were removed under vacuum. After drying, ((prop-2-alkynes-1-yloxy) carbonyl) glycine was obtained. (3.97 g, 25.3 mmol, 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).

[1364] Step 6b):

[1365] 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)amino)-3-methyl-1-oxobutan-2-yl)carbamate (2.7 g, 4.43 mmol, 1.0 equiv) in DMF (5 mL) was added ((prop-2-yn-1-yloxy)carbonyl)glycine (0.732 g, 4.66 mmol, 1.05 equiv), 1-hydroxy-7-azabenzotriazole (0.664 g, 4.88 mmol, 1.1 equiv), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.935 g, 4.88 mmol, 1.1 equiv). The resulting mixture was stirred at ambient temperature for 1 hour, then added dropwise to water (500 mL) and stirred for an additional 20 minutes. The resulting precipitate was filtered, washed with water, and dried in vacuo. Purification by SiO2 ISCO chromatography (0%-50% methanol / dichloromethane) gave tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-(((prop-2-yn-1-yloxy)carbonyl)amino)acetamido)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (LI-4) (1.52 g, 2.40 mmol, 54%). LCMS: MH+ = 634.6; Rt = 1.97 min (5 min acidic method - Method C). 1H NMR(400MHz,DMSO-d6)δppm 0.76-0.91(m,6H)1.30-1.47(m,11H)1.51-1.73(m,2H)1.87-2.00(m,1H)2.89-3.07(m,2H)3.50(t,J=2.32Hz,1H)3.73 -3.87(m,3H)4.37-4.47(m,3H)4.65(d,J=2.45Hz,2H)5.30(t,J=5.44Hz,1H)5.38(s,2H)5.96(t,J=5.81Hz,1H)6.72(br d,J=8.93Hz,1H)7.25(d,J=8.44Hz,1H)7.45(dd,J=8.25,2.02Hz,1H)7.78(br t,J=5.87Hz,1H)7.87-8.00(m,2H)9.51(s,1H)10.04(s,1H).

[1366] Example 1-5: Synthesis of tert-butyl ((S)-1-(((S)-1-((3-(di(prop-2-yn-1-yl)carbamoyl)-4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amin...

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: R 1 It is a reactive group; L1 is a bridging spacer; Lp is a divalent peptide spacer; G-L2-A is a suicide spacer; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D; L3 is the spacer portion; 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 of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: R 1 It is a reactive group; L1 is a bridging spacer; Lp is a bivalent peptide spacer containing one to four amino acid residues; Said The group is selected from: in The * indicates the point of attachment to the N or O of the drug moiety, The *** indicates the attachment point with Lp; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D; L3 is the spacer portion; 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. A compound having the formula (II) or a pharmaceutically acceptable salt thereof, which is a compound having the structure of the formula (II) or a pharmaceutically acceptable salt thereof, in: R 1 It is a reactive group; L1 is a bridging spacer; Lp is a bivalent peptide spacer containing one to four amino acid residues; R 2 It is the hydrophilic part; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D; L3 is the spacer portion; 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 or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: R 1 is -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, L1 is *-C(=O)(CH2) m O(CH2) m -*; *-C(=O)((CH2) m O) t (CH2) n -*; *-C(=O)(CH2) m -*; *-C(=O)NH((CH2) m O) t (CH2) n -**; *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -**; *-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 X1(CH2) m -**; *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -**;*-C(=O)(CH2) m NHC(=O)(CH2) n -**; *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -**; *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -**;*-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, polysarcosine, or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; Each R 3 Independently selected from H and C1-C6 alkyl; R 4 is 2-pyridyl or 4-pyridyl; Each R 5 independently selected from H, C1-C6 alkyl, F, Cl, and -OH; Each R 6 Independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2 and -OH; Each R 7 Independently selected from H, C 1-6 alkyl, fluorine, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C(=O) 1-4 Alkoxy and C substituted by -C(=O)OH 1-4 alkyl; X1 is 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 comprising one to four amino acid residues independently selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said 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)-**、-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 and wherein the ** of said 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-**, -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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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.

5. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: R1 is -ONH2, 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 * 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 selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G; 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)-**、-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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said 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.

6. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: R 1 yes 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 * 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 selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G; 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)-**、-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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said 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.

7. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein: R 1 yes 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 * 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 selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G; 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 )-**, -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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; 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.

8. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein: R 1 yes 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 * 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 selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G; 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, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; 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.

9. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein: R 1 yes 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 * 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 selected from A divalent peptide spacer of , wherein the * of Lp indicates the point of attachment to L1 and the ** of Lp indicates the point of attachment to the -NH- group of G; 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, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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.

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, which has the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

11. A compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

12. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure: wherein R is H, -CH3 or -CH2CH2C(=O)OH.

13. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

14. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure: wherein each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

15. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure: wherein Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R is independently H, -CH3 or -CH2CH2C(=O)OH.

16. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure: in R is H, -CH3 or -CH2CH2C(=O)OH.

17. A compound of formula (I) or formula (II) 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.

18. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure:

19. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure:

20. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure:

21. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure:

22. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof having the structure:

23. A linker having a structure of formula (V), in L1 is a bridging spacer; Lp is a divalent peptide spacer; G-L2-A is a suicide spacer; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 and wherein the ** of A indicates the point of attachment to L2, and L3 is the spacer moiety.

24. The connector of claim 23, wherein: L1 is a bridging spacer; Lp is a bivalent peptide spacer containing one to four amino acid residues; G-L2-A is a suicide spacer; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 and wherein the ** of A indicates the point of attachment to L2, and L3 is the spacer moiety.

25. A joint as claimed in claim 23 or claim 24, wherein: L1 is a bridging spacer; Lp is a bivalent peptide spacer containing one to four amino acid residues; Said The group is selected from: in The * indicates the point of attachment to the N or O of the drug moiety, The *** indicates the attachment point with Lp; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 and wherein the ** of A indicates the point of attachment to L2, and L3 is the spacer moiety.

26. The linker of any one of claims 23 to 25, having a structure of formula (VI), in L1 is a bridging spacer; Lp is a divalent peptide spacer; R 2 It is the hydrophilic part; 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, and L3 is the spacer moiety.

27. A joint as claimed in any one of claims 23 to 26, wherein: L1 is a bridging spacer; Lp is a bivalent peptide spacer containing one to four amino acid residues; R 2 It is the hydrophilic part; 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, and L3 is the spacer moiety.

28. A joint as claimed in any one of claims 23 to 27, wherein: L1 is *-C(=O)(CH2) m O(CH2) m -*; *-C(=O)((CH2) m O) t (CH2) n -*; *-C(=O)(CH2) m -*; *-C(=O)NH((CH2) m O) t (CH2) n -*; *-C(=O)O(CH2) m SSC(R 3 )2(CH2) m C(=O)NR 3 (CH2) m NR 3 C(=O)(CH2) m -*; *-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 X1(CH2) m -*; *-C(=O)((CH2) m O) t (CH2) n X1(CH2) n -*; *-C(=O)(CH2) m NHC(=O)(CH2) n -*; *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n -*; *-C(=O)(CH2) m NHC(=O)(CH2) n X1(CH2) n -*; *-C(=O)((CH2) m O) t (CH2) n NHC(=O)(CH2) n X1(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 selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; Each R 3 Independently selected from H and C1-C6 alkyl; X1 is 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 comprising one to four amino acid residues independently selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine; 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; 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)-**、-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 and wherein the ** of said 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-**, -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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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.

29. A joint as claimed in any one of claims 23 to 28, wherein: 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 * 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 selected from A divalent peptide spacer of , 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)-**、-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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl 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 the 2 Attachment point; R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; and 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.

30. A joint as claimed in any one of claims 23 to 29, wherein: 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 * 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 selected from A divalent peptide spacer of , 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)-**、-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-, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl 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 the 2 Attachment point; R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; and 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.

31. A joint as claimed in any one of claims 23 to 30, wherein: 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 * 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 selected from A divalent peptide spacer of , 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 )-**, -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-**, wherein each R b independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and wherein the ** of said 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 the 2 Attachment point; R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; and A is a bond or -OC(=O)-.

32. A joint as claimed in any one of claims 23 to 31, wherein: 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 * 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 selected from A divalent peptide spacer of , 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, C1-C6 alkyl or C3-C8 cycloalkyl 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 the 2 Attachment point; R 2 is selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polysarcosine, polypeptide or 1 to 3 The hydrophilic part of the C2-C6 alkyl group substituted; and A is a bond or -OC(=O)-.

33. A joint as claimed in any one of claims 23 to 32, wherein: 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 * 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 selected from A divalent peptide spacer of , 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, C1-C6 alkyl or C3-C8 cycloalkyl 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 the 2 Attachment point; R 2 It is polyethylene glycol; and A is a bond or -OC(=O)-.

34. A joint as claimed in any one of claims 23 to 33, having the structure: in R is H, -CH3 or -CH2CH2C(=O)OH.

35. A joint as claimed in any one of claims 23 to 33, having the structure: in R is H, -CH3 or -CH2CH2C(=O)OH.

36. A joint as claimed in any one of claims 23 to 33 having the structure: in R is H, -CH3 or -CH2CH2C(=O)OH.

37. A joint as claimed in any one of claims 23 to 33 having the structure: in Each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

38. A joint as claimed in any one of claims 23 to 33 having the structure: in Each R is independently selected from H, -CH3 or -CH2CH2C(=O)OH.

39. A joint as claimed in any one of claims 23 to 33 having the structure: in Xa is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

40. A joint as claimed in any one of claims 23 to 33 having the structure: in R is H, -CH3 or -CH2CH2C(=O)OH.

41. A joint as claimed in any one of claims 23 to 33 having the structure: in Xb is -CH2-, -OCH2-, -NHCH2-, or -NRCH2- and each R is independently H, -CH3, or -CH2CH2C(=O)OH.

42. A joint as claimed in any one of claims 23 to 33 having the structure:

43. A joint as claimed in any one of claims 23 to 33 having the structure:

44. A joint as claimed in any one of claims 23 to 33 having the structure:

45. A joint as claimed in any one of claims 23 to 33 having the structure:

46. ​​A joint as claimed in any one of claims 23 to 33 having the structure:

47. A conjugate having formula (III): in: Ab is an antibody or a fragment thereof; R 100 is a coupling group; L1 is a bridging spacer; Lp is a divalent peptide linker; G-L2-A is a suicide spacer; R 2 It is the hydrophilic part; L2 is a bond, methylene, neopentylene or C2-C3 alkenylene; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D; L3 is the spacer portion; 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.

48. A conjugate having formula (IV): in: Ab is an antibody or a fragment thereof; R 100 is a coupling group; L1 is a bridging spacer; Lp is a bivalent peptide linker containing one to four amino acid residues; R 2 It is the hydrophilic part; 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 are independently selected from H, C1-C6 alkyl or C3-C8 cycloalkyl and the * of said A indicates the point of attachment to D; L3 is the spacer portion; 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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