Mucin-1 specific antibody conjugates and methods of use thereof

Abstract

The present disclosure provides antibody conjugates (e.g., antibody-drug conjugates (ADCs)) specific for MUC1. The present disclosure also encompasses methods of making such conjugates, as well as methods of using the same. Compositions comprising the ADCs of the present disclosure, including in some instances pharmaceutical compositions, are also provided. In certain aspects, methods of using an ADC are provided that comprise administering a therapeutically effective amount of an ADC of the present disclosure to an individual having a cell proliferative disorder.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 227,666, filed July 30, 2021, U.S. Provisional Application No. 63 / 322,914, filed March 23, 2022, and U.S. Provisional Application No. 63 / 344,932, filed May 23, 2022, the disclosures of each of which are incorporated herein by reference.

[0002] Incorporation by Reference of Electronic Submissions The Sequence Listing is provided herewith as Sequence Listing XML, "RDWD-045WO_SEQ_LIST," having a size of 164 KB, created on October 13, 2022. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] The field of protein-small molecule therapeutic conjugates has made great progress, providing many clinically beneficial drugs with the promise of more in the coming years. Protein-conjugate therapeutics can offer several advantages, for example, due to their specificity, multifunctionality, and relatively low off-target activity, resulting in fewer side effects. Chemical modification of proteins can extend these advantages by making them more potent, stable, or multimodal.

[0004] A number of standard chemical transformations are commonly used to create and manipulate post-translational modifications of proteins. Several methods exist that allow the side chains of specific amino acids to be selectively modified. For example, carboxylic acid side chains (aspartate and glutamate) can be targeted by initial activation with water-soluble carbodiimide reagents followed by reaction with amines. Similarly, lysines can be targeted by the use of activated esters or isothiocyanates, and cysteine ​​thiols can be targeted with maleimides and α-halo-carbonyls.

[0005] One significant obstacle to the creation of chemically modified protein therapeutics or reagents is the production of biologically active, homogeneous forms of the protein. Conjugation of drugs or detectable labels to polypeptides can be difficult to control, resulting in heterogeneous mixtures of conjugates that vary in the number of drug molecules attached and the location of chemical conjugation. In some instances, it may be desirable to use the tools of synthetic organic chemistry to control the conjugation site and / or the drug or detectable label conjugated to the polypeptide to direct the precise and selective formation of chemical bonds on the polypeptide.

[0006] Mucin-1 (also called mucin 1 or MUC1) is a member of the mucin family. Mucins are O-glycosylated proteins that play an essential role in forming a protective mucosal barrier on epithelial surfaces. MUC1 is expressed on the apical membrane of epithelial cells lining the mucosal surfaces of many different tissues, including the lung, breast, stomach, and pancreas. The protein is proteolytically cleaved into an α subunit and a β subunit that form a heterodimeric complex. The N-terminal α subunit functions in cell adhesion, and the C-terminal β subunit is involved in cell signaling. Overexpression, abnormal subcellular localization, and altered glycosylation of this protein are associated with carcinoma. Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need in the art for safe and effective agents that target MUC1 for the treatment of MUC1-associated conditions, such as cancer. [Means for solving the problem]

[0008] The present disclosure provides antibody conjugates (e.g., antibody-drug conjugates (ADCs)) specific for MUC1. The disclosure also encompasses methods of making such conjugates, as well as methods of using them. Each embodiment is described in more detail in the following sections. Compositions, including in some instances pharmaceutical compositions, comprising the ADCs of the present disclosure are also provided. In certain aspects, methods of using an ADC are provided, comprising administering a therapeutically effective amount of an ADC of the present disclosure to an individual with a cell proliferative disorder. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows that the anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, are greater than 99%, greater than 99%, and greater than 98% monomeric, respectively, as determined by size exclusion chromatography (SEC). [Figure 2A] FIG. 1 shows that the anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, bind to recombinant 20-mer MUC1 glycosylated biotin but not to recombinant 60-mer MUC1 non-glycosylated biotin or decoy peptide, as assessed by ELISA. [Figure 2B] FIG. 1 shows that the anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, bind to recombinant 20-mer MUC1 glycosylated biotin but not to recombinant 60-mer MUC1 non-glycosylated biotin or decoy peptide, as assessed by ELISA. [Figure 2C] FIG. 1 shows that the anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, bind to recombinant 20-mer MUC1 glycosylated biotin but not to recombinant 60-mer MUC1 non-glycosylated biotin or decoy peptide, as assessed by ELISA. [Figure 3A]FIG. 1 shows the level of binding by anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, to uncoated streptavidin or Maxisorp plates. [Figure 3B] FIG. 1 shows the level of binding by anti-MUC1 antibodies, MUC1 gB06, MUC1 G12, and MUC1 H02, to uncoated streptavidin or Maxisorp plates. [Figure 4] FIG. 1 shows overlaid histograms showing binding of the indicated antibodies to the indicated cell lines tested in triplicate. [Figure 5] FIG. 1 shows staggered histograms demonstrating binding of the indicated antibodies to the indicated cell lines. [Figure 6] FIG. 1 shows the melting temperatures of the CH2 and Fab regions of the B06, G12, and H02 anti-MUC1 antibodies as determined by differential scanning fluorimetry. [Figure 7] Diagram of aldehyde-labeled antibody production and ADC generation using HIPS-mediated conjugation. (A) A formylglycine recognition sequence (CXPXR) is genetically encoded in the antibody. (B) Cotranslational formylglycine-generating enzyme converts a cysteine ​​within the recognition sequence to a formylglycine residue containing an aldehyde functionality that can be specifically conjugated with (C) a hydrazino-iso-Picteth-Spengler (HIPS) conjugation element. [Figure 8] FIG. 1 shows that CT-tagged B06 antibody conjugated to RED-601 yields a DAR (drug-antibody ratio) of 1.85 as determined by HIC. [Figure 9] FIG. 1 shows that CT-tagged B06 antibody conjugated to RED-601 is 99.3% monomeric as determined by SEC. [Figure 10] FIG. 1 shows that CT-tagged G12 antibody conjugated to RED-601 yields a DAR of 1.89 as determined by HIC. [Figure 11]FIG. 1 shows that CT-tagged G12 antibody conjugated to RED-601 is 99.9% monomeric as determined by SEC. [Figure 12] FIG. 1 shows that CT-tagged H02 antibody conjugated to RED-601 yields a DAR of 1.90 as determined by HIC. [Figure 13] FIG. 1 shows that CT-tagged H02 antibody conjugated to RED-601 is 99.1% monomeric as determined by SEC. [Figure 14]

[0023] Figure 1 depicts the in vitro potency of monomethyl auristatin E (MMAE)-conjugated anti-MUC1 ADCs made from B06, G12, or H02 variant antibodies on T47D cells. Free MMAE is included as a benchmark for payload potency. [Figure 15] In vivo efficacy of MMAE-conjugated anti-MUC1 ADCs carrying an MMAE payload—B06 RED-601 and H02 RED-601—against T47D xenografts. n=10 mice / group; dosing is indicated by arrows. [Figure 16]

[0023] Figure 1 shows representative data demonstrating that the B06 ADC binds to primary human ovarian adenocarcinoma. Four ovarian adenocarcinoma specimens were reacted with the B06 ADC (top row) or an isotype control ADC (bottom row). Brown indicates ADC binding. [Figure 17]

[0023] Figure 1 shows representative data demonstrating that the B06 ADC binds to primary human lung tumors. Four lung cancer specimens were reacted with the B06 ADC (top row) or an isotype control ADC (bottom row). Adenocarcinomas are the left two rows; squamous cell carcinomas are the right two rows. Brown indicates ADC binding. [Figure 18]

[0023] Figure 1 shows representative data demonstrating that the B06 ADC binds to primary human breast tumors. Four breast ductal carcinoma specimens were reacted with the B06 ADC (top row) or an isotype control ADC (bottom row). Brown indicates ADC binding. [Figure 19]Figure 1 shows that the B06 ADC strongly binds to patient-derived xenograft (PDX) tumor models. Four Charles River Laboratories PDX specimens were reacted with the B06 ADC (top row) or an isotype control ADC (bottom row). Brown indicates ADC binding. From left to right, tumor origins are: stomach, breast, lung, and stomach. [Figure 20] FIG. 1 is a diagram of the structure of RED-601, a linker-payload conjugated to an anti-MUC1 antibody (see compound 8 in Example 2). [Figure 21A]

[0023] Figure 1 shows a site map showing possible modification sites for generating aldehyde-tagged Ig polypeptides. The top sequence is the amino acid sequence of a conserved region of an IgG1 light chain polypeptide (SEQ ID NO: 74) showing possible modification sites in the Ig light chain. The bottom sequence is the amino acid sequence of a conserved region of an Ig heavy chain polypeptide (SEQ ID NO: 75) (GenBank Accession No. AAG00909) showing possible modification sites in the Ig heavy chain. The numbering of the heavy and light chains is based on the full-length heavy and light chains. [Figure 21B] FIG. 1 shows an alignment of Homo sapiens immunoglobulin heavy chain constant regions for IgG1 (SEQ ID NO: 47; GenBank P01857.1), IgG2 (SEQ ID NO: 48; GenBank P01859.2), IgG3 (SEQ ID NO: 49; GenBank P01860.2), IgG4 (SEQ ID NO: 50; GenBank AAB59394.1), and IgA (SEQ ID NO: 51; GenBank AAAT74070), depicting modification sites at which an aldehyde tag can be provided on the immunoglobulin heavy chain. [Figure 21C]

[0023] Figure 1 shows an alignment of the Homo sapiens immunoglobulin heavy chain constant regions of IgG1 (SEQ ID NO: 47; GenBank P01857.1), IgG2 (SEQ ID NO: 48; GenBank P01859.2), IgG3 (SEQ ID NO: 49; GenBank P01860.2), IgG4 (SEQ ID NO: 50; GenBank AAB59394.1), and IgA (SEQ ID NO: 51; GenBank AAAT74070), depicting modification sites at which an aldehyde tag can be provided on the immunoglobulin heavy chain. The numbering of the heavy and light chains is based on the complete heavy and light chains. [Figure 21D]

[0023] Figure 1 shows an alignment of immunoglobulin light chain constant regions, showing modification sites that can provide aldehyde tags in the immunoglobulin light chain. Seq1 = Homo sapiens kappa light chain constant region; GenBank CAA75031.1; SEQ ID NO: 52. Seq2 = Homo sapiens kappa light chain constant region; GenBank BAC0168.1; SEQ ID NO: 53. Seq3 = Homo sapiens lambda light chain constant region; GenBank CAA75033; SEQ ID NO: 54. Seq4 = Mus musculus light chain constant region; GenBank AAB09710.1; SEQ ID NO: 55. Seq5 = Rattus norvegicus light chain constant region; GenBank AAD10133; SEQ ID NO: 56. [Figure 22] FIG. 10: Binding of anti-MUC1 affinity matured variant B06 and comparison antibodies, PankoMab and 1B2, to a 20-mer MUC1 glycosylated biotin peptide as assessed by ELISA. [Figure 23] FIG. 10: Binding of anti-MUC1 affinity matured variants G12 and H02 to the 20-mer MUC1 glycosylated biotin peptide as assessed by ELISA. [Figure 24] FIG. 10: Binding of anti-MUC1 affinity matured variant B06 and comparison antibodies, PankoMab and 1B2, to the 60-mer MUC1 non-glycosylated biotin peptide as assessed by ELISA. [Figure 25]FIG. 10: Binding of anti-MUC1 affinity matured variants G12 and H02 to the 60-mer MUC1 non-glycosylated biotin peptide as assessed by ELISA. [Figure 26] FIG. 10: Binding of anti-MUC1 affinity matured variants and parental antibodies to antigen-positive T47D cells or antigen-negative HEK cells assessed by flow cytometry. [Figure 27] 1A-C show in vitro potency of maytansine- or monomethyl auristatin E (MMAE)-conjugated anti-MUC1 ADCs generated from B06 or H02 variant antibodies on UACC-812 cells. Free maytansine was included as a benchmark for payload potency. [Figure 28] FIG. 1 shows that the single tag B06 antibody conjugated to a branched MMAE linker-payload at 91N was 96.4% monomeric as determined by SEC. [Figure 29] FIG. 10 shows that single tag B06 antibody 91N-conjugated to compound 8 yields a DAR of 1.78 as determined by HIC. [Figure 30] FIG. 10 shows that single tag B06 antibody conjugated to compound 8 at 91N is 96.2% monomeric as determined by SEC. [Figure 31] FIG. 1 shows that single tag B06 antibody conjugated to compound 21 at 91N results in a DAR of 3.74 as determined by PLRP. [Figure 32] FIG. 1 shows that single tag B06 antibody conjugated to compound 21 at 91N is 95.9% monomeric as determined by SEC. [Figure 33] FIG. 1 shows that dual-tagged B06 antibody conjugated to compound 21 yields a DAR of 7.47 as determined by PLRP. [Figure 34] FIG. 1 shows that dual-tagged B06 antibody conjugated to compound 21 is 96.7% monomeric as determined by SEC. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, e.g., 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, straight-chain and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0011] The term "substituted alkyl" refers to any alkyl group in which one or more carbon atoms in the alkyl chain (excluding the C1 carbon atom) are substituted with -O-, -N-, -S-, -S(O), n - (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl), and the like may be substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R band R′ and R″, which may be the same or different, are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0012] "Alkylene" is preferably either straight-chain or branched, and includes -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, by way of example, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), iso-propylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0013] "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with a substituent as described for carbon in the definition of "substituted" below.

[0014] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0015] The terms "alkylaminoalkyl," "alkylaminoalkenyl," and "alkylaminoalkynyl" refer to the group R'NHR"-, where R' is an alkyl group as defined herein and R" is an alkylene, alkenylene, or alkynylene group as defined herein.

[0016] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0017] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0018] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0019] The term "alkoxyamino" refers to an -NH-alkoxy group, where alkoxy is defined herein.

[0020] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group has been replaced with a halo group and includes, by way of example, groups such as trifluoromethoxy.

[0021] The term "haloalkyl" refers to a substituted alkyl group as defined above in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0022] The term "alkylalkoxy" refers to -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0023] The term "alkylthioalkoxy" refers to -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0024] "Alkenyl" refers to straight or branched chain hydrocarbyl groups having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1, and preferably 1 to 2, sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0025] The term "substituted alkenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0026] "Alkynyl" refers to a straight-chain or branched monovalent hydrocarbyl group having two to six carbon atoms, preferably two to three carbon atoms, and having at least one, and preferably one or two, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0027] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0028] "Alkynyloxy" refers to the group -O-alkynyl, where alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0029] "Acyl" refers to the groups HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0030] "Acylamino" refers to the group -NR 20 C(O) alkyl, -NR 20 C(O) substituted alkyl, NR 20 C(O)cycloalkyl, -NR 20 C(O)-substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O) substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O) substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O) substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O) heterocycle, and -NR 20 C(O)-substituted heterocycle, wherein R 20is hydrogen or alkyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0031] The term "aminocarbonyl" or "aminoacyl" refers to the group -C(O)NR 51 R 52 In the formula, R 51 and R 52 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; 51 and R 52 may be joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are defined herein.

[0032] "Aminocarbonylamino" is the group -NR 51 C(O)NR 52 R 53 In the formula, R 51 , R 52 and R 53 are independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups are joined to form a heterocyclyl group.

[0033] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0034] The term "acyloxy" refers to alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- groups, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0035] "Aminosulfonyl" refers to the group -SO2NR 51 R 52 In the formula, R 51 and R 52 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 51 and R 52 may be taken together with the nitrogen to which they are attached to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0036] "Sulfonylamino" refers to the group NR 51 SO2R 52 In the formula, R 51 and R 52is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 51 and R 52 may be taken together with the atoms to which they are bonded to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0037] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (e.g., present in a phenyl group) or a ring system having multiple fused rings, which may or may not be aromatic (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), provided that the point of attachment is through an aromatic ring atom. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition of an aryl substituent, such an aryl group is optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0038] "Aryloxy" refers to the group -O-aryl, where aryl is as defined herein, and includes, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups, also defined herein.

[0039] "Amino" refers to the group -NH2.

[0040] The term "substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0041] The term "azido" refers to the group -N3.

[0042] "Carboxyl", "carboxy", or "carboxylate" refers to -CO2H or a salt thereof.

[0043] The terms "carboxyl ester" or "carboxy esters" or "carboxylalkyl" refer to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O refers to -substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocycle, and -C(O)O-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0044] "(Carboxyl ester)oxy" or "carbonate" refers to the groups -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cyclo ... "Alkenyl" refers to alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0045] "Cyano" or "nitrile" refers to the group --CN.

[0046] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0047] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents or from 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0048] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having a single ring or multiple rings and having at least one double bond, preferably 1 to 2 double bonds.

[0049] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0050] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group of from 5 to 10 carbon atoms having mono- or polycyclic rings and having at least one triple bond.

[0051] "Cycloalkoxy" refers to -O-cycloalkyl.

[0052] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0053] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

[0054] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0055] "Heteroaryl" refers to an aromatic group of 1 to 15 carbon atoms, e.g., 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or multiple condensed rings (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl) within the ring system, with at least one ring within the ring system being aromatic. To satisfy valency requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent, such as an alkyl group or other substituent described herein. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of a heteroaryl group may be oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of a heteroaryl substituent, such heteroaryl groups are optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl, and trihalomethyl.

[0056] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0057] "Heteroaryloxy" refers to -O-heteroaryl.

[0058] "Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridges and spiro ring systems, and having 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen; in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of a heterocyclic group can be optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valence requirements, any heteroatom in such a heterocyclic ring can be bonded or unbonded to one or more H or one or more substituents, such as alkyl groups or other substituents described herein.

[0059] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, These include, but are not limited to, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiomorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0060] Unless otherwise constrained by the definition of the heterocycle substituent, such heterocycle groups are optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0061] "Heterocyclyloxy" refers to the group -O-heterocyclyl.

[0062] The term "heterocyclylthio" refers to the group heterocycle-S-.

[0063] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0064] The term "hydroxyamino" refers to the group --NHOH.

[0065] "Nitro" refers to the group -NO2.

[0066] "Oxo" refers to the atom (=O).

[0067] "Sulfonyl" refers to the groups SO-alkyl, SO-substituted alkyl, SO-alkenyl, SO-substituted alkenyl, SO-cycloalkyl, SO-substituted cycloalkyl, SO-cycloalkenyl, SO-substituted cycloalkenyl, SO-aryl, SO-substituted aryl, SO-heteroaryl, SO-substituted heteroaryl, SO-heterocycle, and SO-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. Sulfonyl, by way of example, includes methyl-SO-, phenyl-SO-, and 4-methylphenyl-SO-.

[0068] "Sulfonyloxy" refers to the groups -OSO2-alkyl, OSO2-substituted alkyl, OSO2-alkenyl, OSO2-substituted alkenyl, OSO2-cycloalkyl, OSO2-substituted cycloalkyl, OSO2-cycloalkenyl, OSO2-substituted cycloalkenyl, OSO2-aryl, OSO2-substituted aryl, OSO2-heteroaryl, OSO2-substituted heteroaryl, OSO2-heterocycle, and OSO2-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

[0069] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0070] "Thiol" refers to the group --SH.

[0071] The term "thioxo" or "thioketo" refers to the atom (=S).

[0072] The terms "alkylthio" or "thioalkoxy" refer to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, sulfur can be oxidized to -S(O)-. Sulfoxides can exist as one or more stereoisomers.

[0073] The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

[0074] The term "thioaryloxy" refers to an aryl-S- group, where the aryl group is as defined herein, and includes optionally substituted aryl groups, also defined herein.

[0075] The term "thioheteroaryloxy" refers to the group heteroaryl-S-, where heteroaryl is as defined herein and includes optionally substituted aryl groups, as similarly defined herein.

[0076] The term "thioheterocyclooxy" refers to the group heterocyclyl-S-, where heterocyclyl is as defined herein, and includes optionally substituted heterocyclyl groups, as similarly defined herein.

[0077] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituents, as defined below.

[0078] In addition to the groups disclosed for each individual term herein, substituents to replace one or more hydrogens on a saturated carbon atom in the specified group or radical (any two hydrogens on a single carbon can be replaced by ═O, ═NR, etc.) are also included. 70 , =N-OR 70 , =N2 or =S) are -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 are independently 70 Or, two R 80together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl which may contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have —H or C1-C3 alkyl substitution; and each M + is a counterion with a net positive charge. + are independently, e.g., K + , Na + , Li + Alkaline ions such as + N(R 60 ) 4; or alkaline earth ions, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The "subscript 0.5" means that one of the counterions to such divalent alkaline earth ions may be the ionized form of a compound of the present invention and the other may be a typical counterion such as chloride, or that a doubly ionized compound disclosed herein may serve as the counterion to such divalent alkaline earth ions, or that a doubly ionized compound of the present invention may serve as the counterion to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0079] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkyne, aryl, and heteroaryl group is, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70 、 -SO3 - M + 、 -SO3R 70 、 -OSO2R 70 、 -OSO3 - M + 、 -OSO3R 70 、 -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + 、 -P(O)(OR 70 )2, -C(O)R 70 、 -C(S)R 70 、 -C(NR 70 )R 70 、 -CO2 - M + 、 -CO2R 70 、 -C(S)OR 70 、 -C(O)NR 80 R 80 、 -C(NR 70 )NR 80 R 80 、 -OC(O)R 70 、 -OC(S)R 70 、 -OCO2 - M + 、 -OCO2R 70 、 -OC(S)OR 70 、 -NR 70 C(O)R 70 、 -NR 70 C(S)R 70 、 -NR 70 CO2 - M + 、 -NR 70 CO2R 70 、 -NR 70 C(S)OR 70 、 -NR 70 C(O)NR 80 R 80 、 -NR 70 C(NR 70 )R 70 及び -NR 70 C(NR 70 )NR 80 R 80 であり、式中、R 60 [[ID=***]]、R 70 、R 80 及びM It should be noted that there seems to be some repetition or potential errors in the original text (such as the repeated "R " lines), but the translation is done strictly according to the requirements. If you have any further questions or need corrections, please feel free to let me know.+ is as defined above, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0080] In addition to the groups disclosed for each individual term herein, the substituents of hydrogen on a nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70, -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as defined above.

[0081] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0082] It is understood that for all of the above-defined substituents, polymers arrived at by defining the substituent to have further substituents thereon (e.g., substituted aryls having substituted aryl groups as substituents, which themselves are substituted with substituted aryl groups, which are further substituted with substituted aryl groups, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0083] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is obtained by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0084] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not contain substitutions or patterns of substitution that are sterically impractical and / or synthetically infeasible. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0085] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which can be derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, also includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0086] By "salt thereof" is meant a compound in which a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds not intended for administration to a patient. By way of example, salts of the compounds of the invention include those in which the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0087] "Solvate" refers to a complex formed by the combination of solvent molecules with solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0088] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atomic connectivity but different arrangements of their atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0089] "Tautomers" refers to alternative forms of molecules that differ only in the position of the atoms' electron bonding and / or protons, e.g., enol-keto and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing the -N=C(H)-NH- ring atom arrangement, e.g., pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One skilled in the art will recognize that other tautomeric ring atom arrangements are possible.

[0090] It will be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include all permutations of salts, solvates, and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

[0091] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer composed of two dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole-chain or single-chain antibodies) that retain specific binding to an antigen, including, but not limited to, Fab fragments, Fv fragments, scFv fragments, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies can be further conjugated to other moieties, such as a member of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). The antibody may also be bound to a solid support, including, but not limited to, a polystyrene plate or beads. Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to the antigen, and monoclonal antibodies are also encompassed by the term. The antibody may be monovalent or bivalent.

[0092] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. These include diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0093] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRS from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0094] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0095] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0096] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.

[0097] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0098] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, expressed as the dissociation constant (Kd). The affinity can be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold greater than the affinity of an antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0099] The term "binding" refers to a direct association between two molecules, e.g., by ionic and / or hydrogen bonding interactions, including covalent, electrostatic, hydrophobic, and salt and water bridge interactions. A subject anti-MUC1 antibody specifically binds to an epitope within a MUC1 polypeptide, e.g., a human MUC1 polypeptide, e.g., glycosylated MUC1 or a fragment thereof. Non-specific binding occurs within about 10 -7 Binding with an affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0100] The term "specifically binds" in reference to antibodies and antigens means that the antibody binds to the antigen at a specific concentration, e.g., about 10 5 M -1 or higher affinity or K a (i.e., the equilibrium association constant for a specific binding interaction with units of 1 / M).

[0101] "High affinity" binding is defined as binding with a 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or K exceeding it a Alternatively, affinity is expressed in M ​​units (e.g., 10 -5 M~10 -13 Equilibrium dissociation constant (K M) of a specific binding interaction D In some embodiments, specific binding can be defined as the binding of an antibody to a cell surface area greater than or equal to about 10 -5 M, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M, about 10 -10M or less, about 10 -11 M or less, or about 10 -12 K below M D The binding affinity of an antibody to an antigen can be readily determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., a BIAcore 2000 instrument using the general procedures outlined by the manufacturer); by radioimmunoassay; or others.

[0102] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. [Table 1] Throughout this disclosure, the numbering of immunoglobulin heavy chain and immunoglobulin light chain residues is as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference.

[0103] As used herein, the term "framework" when used in reference to an antibody variable region is intended to refer to all amino acid residues outside the CDR regions within the variable region of an antibody. Variable region frameworks are generally discontinuous amino acid sequences between about 100 and 120 amino acids in length, but are intended to refer only to amino acids outside the CDRs. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs.

[0104] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region or may comprise a constant region with pre-existing amino acid sequence modifications (e.g., additions, deletions, and / or substitutions, etc.).

[0105] In the context of Ig polypeptides, the term "constant region" is well understood in the art and refers to the C-terminal region of an Ig heavy chain or an Ig light chain. The Ig heavy chain constant region comprises CH1, CH2, and CH3 domains (and a CH4 domain, where the heavy chain is a μ or ε heavy chain). In naturally occurring Ig heavy chains, the CH1, CH2, CH3 (and CH4, if present) domains begin immediately (C-terminally) after the heavy chain variable (VH) region and are each about 100 to 130 amino acids in length. In naturally occurring Ig light chains, the constant region begins immediately (C-terminally) after the light chain variable (VL) region and is about 100 to 120 amino acids in length.

[0106] An "epitope" is a site on an antigen (e.g., a site on MUC1) to which an antibody binds. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by protein folding (e.g., tertiary folding). Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more usually at least five or eight to ten amino acids in a linear or spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Several commercial laboratories offer epitope mapping services. Epitopes bound by antibodies immunoreactive with membrane-associated antigens can be present on the surface of a cell (e.g., the extracellular region of a transmembrane protein), and as a result, such epitopes are considered to be cell surface accessible, solvent accessible, and / or cell surface exposed.

[0107] "Genetically encodable," as used in reference to an amino acid sequence of a polypeptide, peptide, or protein, means that the amino acid sequence is composed of amino acid residues that can be produced by transcription and translation of a nucleic acid encoding the amino acid sequence, and the transcription and / or translation can occur in a cell or in a cell-free in vitro transcription / translation system.

[0108] The term "control sequence" refers to DNA sequences that facilitate the expression of an operably linked coding sequence in a particular expression system, such as mammalian cells, bacterial cells, cell-free synthesis, etc. Control sequences suitable for prokaryotic systems may include, for example, a promoter, operator sequence, and ribosome binding site. Eukaryotic systems may utilize promoters, polyadenylation signals, and enhancers.

[0109] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation initiation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation or amplification reactions. Synthetic oligonucleotide adapters or linkers can be used to join sequences according to conventional practice.

[0110] As used herein, the term "expression cassette" refers to a segment of nucleic acid, usually DNA, that can be inserted into a nucleic acid (e.g., by use of restriction sites compatible for ligation into a construct of interest, or by homologous recombination into the construct of interest or into a host cell genome). Generally, the nucleic acid segment comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. The expression cassette can also include elements that facilitate expression of the polynucleotide encoding the polypeptide of interest in a host cell, e.g., a mammalian host cell. These elements can include, but are not limited to, a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[0111] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 90%, 95%, or 98%, e.g., greater than 99%, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In some examples, isolated antibodies will be prepared by at least one purification step.

[0112] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are paired and produced by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, and serum are examples of tissues that produce natural antibodies. For example, an antibody produced by antibody-producing cells isolated from a first animal immunized with an antigen is a natural antibody.

[0113] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody containing one or more amino acids (e.g., in the framework region, constant region, or CDR) substituted with a correspondingly positioned amino acid from a human antibody. Generally, a humanized antibody generates a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting, veneering or resurfacing, chain shuffling, etc. In certain embodiments, framework substitutions are identified by modeling the interactions between CDR and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at specific positions. Thus, the above-described antibodies can be humanized using methods well known in the art.

[0114] In certain embodiments, the antibody molecules disclosed herein comprise a heavy chain comprising a variable heavy chain region as provided herein and a human IgG1 constant region having the amino acid sequence set forth in UniProt:P01857-1, version 1. In certain embodiments, the antibody molecules disclosed herein comprise a light chain comprising a variable light chain region as provided herein and a human light chain constant region. In certain embodiments, the human light chain constant region is a human kappa light chain constant region having the amino acid sequence set forth in UniProtKB / Swiss-Prot:P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in a subject antibody may comprise mutations, such as substitutions, to modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody-dependent cellular cytotoxicity (ADCC). The numbering of substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is commonly used to refer to residues in immunoglobulin heavy chain constant regions (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0115] The term "chimeric antibody" refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody may be linked to human constant segments such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of variable or antigen-binding domains from a mouse antibody and constant or effector domains from a human antibody, although domains from other mammalian species may also be used.

[0116] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless otherwise specified, "polypeptide," "peptide," and "protein" can include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms also include, but are not limited to, fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells); immunotagged proteins; and the like. In the context of antibodies, it is clear that a chain or domain includes a polypeptide.

[0117] "Native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein and refer to the amino acid sequence of a polypeptide before it is modified to include modified amino acid residues.

[0118] The terms "amino acid analog," "unnatural amino acid," and the like, may be used interchangeably and include amino acid-like compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some examples, amino acid analogs share the backbone and / or side chain structure of one or more naturally occurring amino acids, with the difference(s) being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl) or atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (such as a single bond, such as a double bond), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids, etc.

[0119] Terms such as "amino acid side chain" or "side chain of an amino acid" can be used to refer to a substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. Amino acid side chains can also include amino acid side chains as described in connection with modified amino acids and / or conjugates described herein.

[0120] The term "conjugate" generally refers to a covalent or non-covalent, usually covalent, chemical bond that proximally associates one molecule of interest with a second molecule of interest. In some embodiments, the agent is selected from a half-life extending moiety, a labeling agent, and a therapeutic agent. For half-life extension, for example, antibodies of the present disclosure may optionally be modified to provide an improved pharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, etc.). Modifications that can improve serum half-life are of interest.

[0121] Terms such as "carbohydrate" may be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides, etc. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest have been substituted (substituted with any convenient substituent), modified (converted to another group using any convenient chemistry), or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the subject compounds and conjugates.

[0122] As used herein, the term "isolated" is meant to describe a compound of interest in an environment that is different from the environment that the compound naturally occurs in. "Isolated" is meant to include compounds within a sample in which the compound of interest is substantially enriched and / or in which the compound of interest has been partially or substantially purified.

[0123] As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free from other components with which it is naturally associated.

[0124] The term "physiological conditions" is meant to encompass conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0125] "Reactive partner" refers to a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include the cysteine ​​or serine of a sulfatase motif and formylglycine generating enzyme (FGE), which react to form a reaction product of a converted aldehyde tag that contains formylglycine (fGly) in place of the cysteine ​​or serine in the motif. Other exemplary reactive partners include the aldehyde of the fGly residue of a converted aldehyde tag (e.g., reactive aldehyde group), and an "aldehyde-reactive reactive partner" that contains an aldehyde-reactive group and a moiety of interest, which reacts to form a reaction product of a polypeptide having a moiety of interest conjugated to the polypeptide via the fGly residue.

[0126] "N-terminus" refers to the terminal amino acid residue of a polypeptide having a free amine group; the amine group in the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0127] "C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group; the carboxyl group in the non-C-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0128] An "internal site" when used in reference to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or C-terminus.

[0129] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, e.g., halting its development, and (c) relieving the disease, e.g., causing regression of the disease.

[0130] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0131] A "therapeutically effective amount" or "effective amount" refers to the amount of a subject anti-MUC1Ab that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the anti-MUC1Ab, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0132] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0133] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the relevant methods and / or materials for which the publications are cited.

[0135] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of such antibodies, a reference to a "CDR" includes a reference to one or more CDRs and equivalents thereof known to those skilled in the art, and so forth. It should further be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0136] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0137] Detailed Description The present disclosure provides antibody conjugates (e.g., antibody-drug conjugates (ADCs)) specific for MUC1. The present disclosure also encompasses methods of making such conjugates, as well as methods of using them. Each embodiment is described in more detail in the following sections. Compositions comprising the ADCs of the present disclosure, including in some instances pharmaceutical compositions, are also provided. In certain aspects, methods of using an ADC are provided, comprising administering a therapeutically effective amount of an ADC of the present disclosure to an individual having a cell proliferative disorder.

[0138] MUC1 antibody and its antibody-drug conjugate As summarized above, the present disclosure provides conjugates of antibodies specific for MUC1 (e.g., antibody-drug conjugates (ADCs)). Additionally, the present disclosure provides anti-MUC1 antibodies that include a fGly residue.

[0139] Antibody-drug conjugates The present disclosure provides conjugates, e.g., antibody-drug conjugates (ADCs) of antibodies specific for MUC1. By "conjugate" is meant that a polypeptide (e.g., an antibody) is covalently attached to a moiety of interest (e.g., a drug or active agent). For example, an antibody-drug conjugate according to the present disclosure comprises one or more drugs or active agents covalently attached to an antibody. In certain embodiments, the polypeptide (e.g., an antibody) and one or more drugs or active agents are attached to each other via one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds can include a linker, such as a cleavable linker described herein.

[0140] In certain embodiments, the conjugate is a polypeptide conjugate comprising a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, the one or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest, including, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilizing the polypeptide on a membrane or surface. In certain embodiments, the conjugate is a drug conjugate and the polypeptide is an antibody, thus providing an antibody-drug conjugate. For example, the conjugate can be a drug conjugate in which the polypeptide is conjugated to one or more drugs or active agents. Various types of drugs and active agents can be used in the conjugates, as described in more detail below.

[0141] One or more drugs or active agents can be conjugated to a polypeptide (e.g., an antibody) at any desired site on the polypeptide. Thus, the present disclosure provides, for example, polypeptides having drugs or active agents conjugated at or near the C-terminus of the polypeptide. Other examples include polypeptides having drugs or active agents conjugated at or near the N-terminus of the polypeptide. Examples also include polypeptides having drugs or active agents conjugated between the C-terminus and N-terminus of the polypeptide (e.g., at an internal site on the polypeptide). Combinations of the above, in which a polypeptide is conjugated to two or more drugs or active agents, are also possible.

[0142] In certain embodiments, a conjugate of the present disclosure comprises one or more drugs or active agents conjugated to an amino acid residue of a polypeptide at the alpha carbon of the amino acid residue. Stated another way, a conjugate comprises a polypeptide in which the side chain of one or more amino acid residues in the polypeptide has been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents via a linker described herein). For example, a conjugate comprises a polypeptide in which the alpha carbon of one or more amino acid residues in the polypeptide has been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents via a linker described herein).

[0143] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more moieties, for example, two, three, four, five, six, seven, eight, nine, or ten or more moieties. A moiety can be conjugated to a polypeptide at one or more sites in the polypeptide. For example, one or more moieties can be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties can be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example, where a polypeptide is conjugated to a first moiety at a first amino acid residue and to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to first and second moieties at a first amino acid residue and to a third and fourth moiety at a second amino acid residue.

[0144] One or more amino acid residues of a polypeptide conjugated to one or more moieties of interest can be naturally occurring amino acids, non-natural amino acids, or a combination thereof. For example, a conjugate can include one or more drugs or active agents conjugated to naturally occurring amino acid residues of the polypeptide. In other examples, a conjugate can include one or more drugs or active agents conjugated to non-natural amino acid residues of the polypeptide. One or more drugs or active agents can be conjugated to the polypeptide at a single natural or non-natural amino acid residue described herein. One or more natural or non-natural amino acid residues in a polypeptide can be conjugated to one or more moieties described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in a polypeptide can each be conjugated to one or two moieties such that multiple sites in the polypeptide are conjugated to a moiety of interest.

[0145] In certain embodiments, the polypeptide (e.g., an antibody) and the moiety of interest (e.g., a drug or active agent) are conjugated via a conjugation moiety. For example, the polypeptide and the moiety of interest can each be bound (e.g., covalently bonded) to a conjugation moiety, thus indirectly linking the polypeptide and the moiety together via the conjugation moiety. Optionally, the conjugation moiety comprises a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. For example, a general scheme for coupling a moiety of interest to a polypeptide via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the general reaction scheme below. Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also referred to herein as hydrazino-iso-pictet-Spengler (HIPS) conjugation moieties and aza-hydrazino-iso-pictet-Spengler (azaHIPS) conjugation moieties, respectively. [ka] In the above reaction scheme, each R comprises a moiety of interest (e.g., a drug or active agent) conjugated to a polypeptide (e.g., conjugated to the polypeptide via a cleavable linker described herein), and n is an integer from 1 to 4. As shown in the above reaction scheme, a polypeptide comprising a 2-formylglycine residue (fGly) is reacted with a drug or active agent modified to include a conjugation moiety (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to produce a polypeptide conjugate attached to the conjugation moiety, thus attaching the drug or active agent to the polypeptide via the conjugation moiety.

[0146] As described herein, a moiety can be any of a variety of moieties, such as, but not limited to, a chemical entity, such as a detectable label, or a drug or active agent. R' and R" can each independently be any desired substituent, including, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z can be CR 61 , N.R. 62 , N, O or S, where R 61 and R 62 are each independently selected from any of the substituents described for R' and R" above.

[0147] As shown in the conjugates and compounds described herein, other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety may be attached (e.g., covalently attached) to a linker. Thus, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to a drug or active agent via a linker. Various embodiments of linkers that can attach the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker as described herein.

[0148] As shown in the conjugates and compounds described herein, additional hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also possible. For example, a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be attached (e.g., covalently attached) to two or more linkers. Thus, embodiments of the present disclosure include hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties attached to two or more drugs or active agents via corresponding linkers, respectively. Thus, conjugates of the present disclosure can include two or more linkers, each linker attaching a corresponding drug or active agent to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. Thus, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety and two or more linkers can be considered collectively as a "branched linker," with the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to two or more "branches," each branch comprising a linker attached to a drug or active agent.

[0149] The same combination of different payloads can be conjugated to a polypeptide via a branched linker. In certain embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are the same payload (e.g., drugs, active agents, or detectable labels). For example, the first branch of the branched linker can be attached to a payload (e.g., a drug, active agent, or detectable label), and the second branch of the branched linker can be attached to the same payload (e.g., a drug, active agent, or detectable label) as the first branch.

[0150] In other embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are different payloads (e.g., drugs, active agents, or detectable labels). For example, a first branch of the branched linker can be attached to a first payload (e.g., a first drug, active agent, or detectable label), and a second branch of the branched linker can be attached to a second payload (e.g., a second drug, active agent, or detectable label) that is different from the first payload (e.g., the first drug, active agent, or detectable label) attached to the first branch.

[0151] In certain embodiments, a polypeptide (e.g., an antibody) can be conjugated to one or more moieties of interest, wherein one or more amino acid residues of the polypeptide are modified prior to conjugation to the moiety of interest. Modification of one or more amino acid residues of the polypeptide can produce a polypeptide containing one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide can contain one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to the moiety of interest (e.g., one or more moieties containing a conjugation moiety such as the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety described above). For example, an amino acid of the polypeptide can be modified to contain a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be contained in an "aldehyde tag" or "ald tag," which, as used herein, refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR (SEQ ID NO: 5)) that has been converted to contain a 2-formylglycine residue (referred to herein as "fGly") by the action of formylglycine generating enzyme (FGE). The fGly residue generated by FGE is sometimes referred to as "formylglycine." Alternatively, the term "aldehyde tag" is used herein to refer to an amino acid sequence that includes a "converted" sulfatase motif (e.g., a sulfatase motif in which a cysteine ​​or serine residue has been converted to fGly by the action of FGE, e.g., L(fGly)TPSR (SEQ ID NO: 24)). A converted sulfatase motif can be produced from an amino acid sequence containing an "unconverted" sulfatase motif (e.g., a sulfatase motif in which the cysteine ​​or serine residue has not been converted to fGly by FGE, but can be converted, e.g., the sequence: L(C / S)TPSR). "Conversion," as used in the context of the action of formylglycine generating enzyme (FGE) on a sulfatase motif, refers to the biochemical modification of a cysteine ​​or serine residue in the sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly, or Ser to fGly).Further aspects of the aldehyde tag and its use in site-specific protein modification are described in US Pat. No. 7,985,783 and US Pat. No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0152] In some cases, a polypeptide containing an fGly residue can be conjugated to a moiety of interest by reaction of fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety described above) to produce a conjugate. For example, an fGly-containing polypeptide can be contacted with a reactive partner-containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some examples, the reactive partner-containing drug can include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety described above. For example, a drug or active agent can be modified to include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, the drug or active agent is covalently attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl via a linker, such as those described in detail herein.

[0153] In certain embodiments, the conjugates of the present disclosure comprise a polypeptide (e.g., an antibody) having at least one amino acid residue conjugated to one or more moieties of interest (e.g., drugs or active agents). To generate the conjugate, the amino acid residue of the polypeptide can be modified and then coupled to one or more drugs or active agents conjugated to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above. In certain embodiments, the amino acid residue of the polypeptide (e.g., an antibody) is a cysteine ​​or serine residue modified to an fGly residue, as described above. In certain embodiments, the modified amino acid residue (e.g., an fGly residue) is conjugated to a drug or active agent containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above, to provide conjugates of the present disclosure in which one or more drugs or active agents are conjugated to the polypeptide via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As used herein, the term fGly' refers to a modified amino acid residue of a polypeptide (eg, an antibody) that is attached to a moiety of interest (eg, a drug or active agent).

[0154] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one amino acid residue attached to a linker described herein, which is then conjugated to one or more drugs or active agents. For example, the conjugate may comprise a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') conjugated to one or more moieties of interest (e.g., one or more drugs or active agents), as described above.

[0155] An embodiment of the present disclosure is a compound of formula (I): [ka] comprising a conjugate of During the ceremony, Z is CR 4 or N, R 1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 may be cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W 1 is a drug, W 2 is an anti-MUC1 antibody.

[0156] In certain embodiments, Z is CR 4 or N, and in certain embodiments, Z is CR 4 In certain embodiments, Z is N.

[0157] In certain embodiments, R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0158] In certain embodiments, R 1 is hydrogen. In certain embodiments, R 1 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 1 is methyl. In certain embodiments, R 1 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 1 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkynyl or substituted alkynyl, such as a substituted alkenyl. 1 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 1 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 1 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 1 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0159] In certain embodiments, R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 may be cyclically linked to form a 5- or 6-membered heterocyclyl.

[0160] In certain embodiments, R 2is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 2 is alkynyl or substituted alkynyl. In certain embodiments, R 2 is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acylamino or aminoacyl. In certain embodiments, R 2 is an alkylamide or a substituted alkylamide. In certain embodiments, R 2 is sulfonyl. In certain embodiments, R2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 2 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 2 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 2 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 2 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0161] In certain embodiments, R 3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 3 is hydrogen. In certain embodiments, R 3is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 3 is methyl. In certain embodiments, R 3 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 3 is alkynyl or substituted alkynyl. In certain embodiments, R 3 is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acylamino or aminoacyl. In certain embodiments, R 3 is an alkylamide or a substituted alkylamide. In certain embodiments, R 3 is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 3 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 3 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 3 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 3 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0162] In certain embodiments, R 2 and R 3 may be cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 6-membered heterocyclyl.

[0163] In certain embodiments, each R 4is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0164] Each R 4 The various possibilities for are described in more detail below. In certain embodiments, R 4 is hydrogen. In certain embodiments, each R 4 is hydrogen. In certain embodiments, R 4 is halogen, for example, F, Cl, Br, or I, and in certain embodiments, R 4 is F, and in certain embodiments, R 4 is Cl. In certain embodiments, R 4 is Br. In certain embodiments, R 4 is I. In certain embodiments, R 4 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 4 is methyl. In certain embodiments, R 4 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 4 is alkynyl or substituted alkynyl. In certain embodiments, R4 is alkoxy or substituted alkoxy. In certain embodiments, R 4 is amino or substituted amino. In certain embodiments, R 4 is carboxyl or carboxyl ester. In certain embodiments, R 4 is acyl or acyloxy. In certain embodiments, R 4 is acylamino or aminoacyl. In certain embodiments, R 4 is an alkylamide or a substituted alkylamide. In certain embodiments, R 4 is sulfonyl. In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 4 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl (e.g., phenyl or substituted phenyl). 4 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 4 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 4 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C3~5 It is a substituted heterocyclyl.

[0165] In certain embodiments, W 1 is a drug. Further description of drugs can be found in the disclosure herein.

[0166] In certain embodiments, W 2 is an anti-MUC1 antibody. In certain embodiments, W 2 comprises one or more fGly' residues as described herein. In certain embodiments, the anti-MUC1 antibody is attached to the remainder of the conjugate via an fGly' residue as described herein. Further description of anti-MUC1 antibodies that find use in the present conjugates is found in the disclosure herein.

[0167] In certain embodiments, the compound of Formula (I) comprises a linker, L. The linker can be utilized to attach a conjugation moiety (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to one or more moieties of interest. The linker can be attached (e.g., covalently attached) to a conjugation moiety (e.g., as described herein) at any convenient position. For example, the linker can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety can be used to conjugate the linker (and thus the drug) to a polypeptide, such as an anti-MUC1 antibody. For example, the conjugation moiety can be used to conjugate the linker (and thus the drug) to a modified amino acid residue of a polypeptide, such as an fGly residue of an anti-MUC1 antibody.

[0168] In certain embodiments, L is selected from the group consisting of a conjugation moiety W 1 and thus the conjugation moiety is linked to W via the linker L. 1 and indirectly binds to W as described above. 1is a drug, and thus L attaches the conjugation moiety to the drug, e.g., the conjugation moiety is indirectly attached to the drug via the linker L.

[0169] Any convenient linker may be utilized in the subject conjugates. In certain embodiments, L comprises a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, L comprises an alkyl group or a substituted alkyl group. In certain embodiments, L comprises an alkenyl or substituted alkenyl group. In certain embodiments, L comprises an alkynyl or substituted alkynyl group. In certain embodiments, L comprises an alkoxy or substituted alkoxy group. In certain embodiments, L comprises an amino group or a substituted amino group. In certain embodiments, L comprises a carboxyl group or a carboxyl ester group. In certain embodiments, L comprises an acylamino group. In certain embodiments, L comprises an alkylamido or substituted alkylamido group. In certain embodiments, L comprises an aryl or substituted aryl group. In certain embodiments, L comprises a heteroaryl or substituted heteroaryl group. In certain embodiments, L comprises a cycloalkyl or substituted cycloalkyl group. In certain embodiments, L comprises a heterocyclyl or substituted heterocyclyl group.

[0170] In certain embodiments, L comprises a polymer. For example, the polymer can include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., when the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated in the conjugates and compounds described in more detail below.

[0171] In some embodiments, L is of the formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - wherein L 1 , L 2 , L 3 , L 4 , L 5 and L 6 are each independently a linker subunit, a, b, c, d, e, and f are each independently 0 or 1, and the total of a, b, c, d, e, and f is 1 to 6.

[0172] In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0. In certain embodiments, a is 1 and b, c, d, e, and f are each 0.

[0173] In certain embodiments, the linker subunit L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 2 When present, is attached to the drug. In certain embodiments, the linker subunit L 3 When present, is attached to the drug. In certain embodiments, the linker subunit L 4 When present, is attached to the drug. In certain embodiments, the linker subunit L 5 When present, is attached to the drug. In certain embodiments, the linker subunit L 6 If present, is bound to the drug.

[0174] Any convenient linker subunit may be utilized in the linker, L. Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 and L 6 Each (when present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0175] In some embodiments, L 1 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 In some embodiments, L 1 comprises an aryl group or a substituted aryl group. In some embodiments, L 1 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0176] In some embodiments, L 2 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L2 In some embodiments, L 2 In some embodiments, L 2 comprises an amino acid residue. In some embodiments, L 2 In some embodiments, L 2 comprises an aryl group or a substituted aryl group. In some embodiments, L 2 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0177] In some embodiments, L 3 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 comprises an amino acid residue. In some embodiments, L 3 In some embodiments, L 3 comprises an aryl group or a substituted aryl group. In some embodiments, L 3 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0178] In some embodiments, L 4 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 comprises an amino acid residue. In some embodiments, L 4In some embodiments, L 4 comprises an aryl group or a substituted aryl group. In some embodiments, L 4 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0179] In some embodiments, L 5 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L 5 comprises an amino acid residue. In some embodiments, L 5 In some embodiments, L 5 comprises an aryl group or a substituted aryl group. In some embodiments, L 5 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0180] In some embodiments, L 6 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 6 In some embodiments, L 6 In some embodiments, L 6 comprises an amino acid residue. In some embodiments, L 6 In some embodiments, L 6 comprises an aryl group or a substituted aryl group. In some embodiments, L 6 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0181] In some embodiments, L is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, wherein -(L 1 ) a -is-(T 1 -V 1 ) a - and -(L 2 ) b -is-(T 2 -V 2 ) b - and -(L 3 ) c -is-(T 3 -V 3 ) c - and -(L 4 ) d -is-(T 4 -V 4 ) d - and -(L 5 ) e -is-(T 5 -V 5 ) e - and -(L 6 ) f -is-(T 6 -V 6 ) f - and In the formula, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 is a tether group, if present, V 1 , V 2 , V 3 , V 4 , V 5 and V6 is a covalent bond or linking functional group, if present; a, b, c, d, e, and f each independently represent 0 or 1, and the total of a, b, c, d, e, and f is 1 to 6.

[0182] As noted above, in certain embodiments, L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, V 1 is attached to the drug. In certain embodiments, L 2 When present, T is bound to the drug. 2 If present, is bound to the drug, or V 2 When present, L is attached to the drug. 3 When present, T is bound to the drug. 3 If present, is bound to the drug, or V 3 When present, L is attached to the drug. 4 When present, T is bound to the drug. 4 If present, is bound to the drug, or V 4 When present, L is attached to the drug. 5 When present, T is bound to the drug. 5 If present, is bound to the drug, or V 5 When present, L is attached to the drug. 6 When present, T is bound to the drug. 6 If present, is bound to the drug, or V 6If present, is bound to the drug.

[0183] Tether group T 1 , T 2 , T 3 , T 4 , T 5 and T 6 Any convenient tether group can be utilized in the subject linker. 1 , T 2 , T 3 , T 4 , T 5 and T 6 are covalent bonds, (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester, wherein each w is an integer of 1 to 20, each n is an integer of 1 to 30, each p is an integer of 1 to 20, and each m is an integer of 1 to 12.

[0184] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) is (C1~C 12 ) alkyl or substituted (C1-C 12In certain embodiments, (C1-C 12 )Alkyl is a straight or branched alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, (C1-C 12 ) Alkyl is alkyl or substituted alkyl, for example C1-C 12 Alkyl or C1-C 10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) alkyl is C2-alkyl. For example, (C1-C 12 ) Alkyl is alkylene or substituted alkylene, for example C1-C 12 Alkylene or C1-C 10 It can be alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some cases, (C1-C 12 ) Alkyl is C2-alkylene (e.g., CH2CH2).

[0185] In certain embodiments, the substitution (C1-C 12 ) alkyl is a straight or branched substituted alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkyl or substituted C1-C 10 It can be alkyl, or substituted alkyl, such as substituted C1-C6 alkyl, or substituted C1-C3 alkyl. In some cases, substituted (C1-C 12 ) alkyl is a substituted C2-alkyl. For example, substituted (C1-C 12 ) alkyl is a substituted C1-C 12 Alkylene or substituted C-C 10It can be alkylene, or substituted alkylene, such as substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some cases, the substituted (C1-C 12 ) Alkyl is a substituted C2-alkylene.

[0186] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some examples, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T 6 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl is (C1-C 12 ) Alkyl, substituted (C1-C 12 ) may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some examples, the substituted aryl is a substituted phenyl and the substituent comprises a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0187] In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) includes heteroaryl or substituted heteroaryl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T6 ) includes cycloalkyl or substituted cycloalkyl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T 6 ) includes heterocyclyl or substituted heterocyclyl. In some examples, the substituent on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl includes a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0188] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises an ethylenediamine (EDA) moiety, e.g., an EDA-containing tether group. In certain embodiments, (EDA) w comprises one or more EDA moieties, e.g., where w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) moieties may be substituted at one or more convenient positions with any convenient substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety has the structure: [ka] , In the formula, y is an integer of 1 to 6, or 0 or 1, and each R 12is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be cyclically linked, for example to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 In certain embodiments, y is 1 and the adjacent R 12 The groups are selected from hydrogen, alkyl (eg, methyl), and substituted alkyl (eg, lower alkyl-OH, such as ethyl-OH or propyl-OH).

[0189] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be substituted at one or more convenient positions with any convenient substituent, such as an alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety has the structure: [ka] In the formula, R 12 is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxy-modified polyethylene glycol.

[0190] In certain embodiments, R 12 is a compound of the formula: (PEG) k which comprises a polyethylene glycol moiety described by the structure: [ka] , where k is an integer from 1 to 20, e.g., from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, k is 2. In certain embodiments, R 17 is selected from OH, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 17is COOH.

[0191] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) is (PEG) n (PEG) n is a polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG) n The structure: [ka] , In the formula, n is an integer from 1 to 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0192] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) is (AA) pwhere AA is an amino acid residue. Any convenient amino acid can be utilized. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary α-amino acids and β-alanine, and non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids. In certain embodiments, p is an integer between 1 and 50, e.g., between 1 and 40, 1 and 30, 1 and 20, 1 and 12, or 1 and 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0193] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6) include amino acid analogs. Amino acid analogs include compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some examples, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, with the difference(s) being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl) or atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (such as a single bond, such as a double bond), or a combination thereof. For example, amino acid analogs may include alpha-hydroxy acids and alpha-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfoalanine, etc.

[0194] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) is the formula -(CR 13 OH) m wherein m is 0 or n is an integer from 1 to 50, e.g., from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12, or from 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R 13is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 13 is hydrogen. In certain embodiments, R 13 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 13 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. In certain embodiments, R 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 13 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 13 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 13 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 13 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0195] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are selected from R 13 is as described above.

[0196] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T6 ) includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP) or para-hydroxy-phenyl (PHP).

[0197] In some embodiments, the tether group has the following structure: [ka] The MABO group is described by:

[0198] In some embodiments, the tether group has the following structure: [ka] This includes the MABC group described by:

[0199] In some embodiments, the tether group has the following structure: [ka] This includes the PABO group described by:

[0200] In some embodiments, the tether group has the following structure: [ka] This includes the PABC group described by:

[0201] In some embodiments, the tether group has the following structure: [ka] The PAB group includes the PAB group described by:

[0202] In some embodiments, the tether group has the following structure: [ka] The PABA group is described by:

[0203] In some embodiments, the tether group has the following structure: [ka] This includes the PAP group described by:

[0204] In some embodiments, the tether group has the following structure: [ka] The PHP group includes the PHP group described by:

[0205] In certain embodiments, each R 14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0206] In certain embodiments, R 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 14 is C 2~6Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 14 is alkynyl or substituted alkynyl. In certain embodiments, R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14 is acyl or acyloxy. In certain embodiments, R 14 is acylamino or aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 14 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 14 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 14 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5In certain embodiments, R 14 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0207] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more further groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0208] In certain embodiments of the linker L, the tether group T 1 , T 2 , T 3 , T 4 , T 5 or T 6 may each be substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0209] In certain embodiments, the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0210] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] may be selected from:

[0211] Linking functional group V 1 , V 2 , V 3 , V 4 , V 5 and V 6 With respect to V, any convenient linking functional group may be utilized for the linker L. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoridate, and the like. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 and V 6 is a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 is independently selected from SO2- and -P(O)OH-, and q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0212] In some embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0213] In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 15 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 15 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 15 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 15 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 15 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0214] In certain embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are independently selected from R 15 is as described above.

[0215] In certain embodiments, the tether group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises a hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

[0216] As noted above, in some embodiments, L is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) fwherein a, b, c, d, e, and f each independently represent 0 or 1, and the total of a, b, c, d, e, and f is 1 to 6.

[0217] In some embodiments, in the linker L: T 1 is (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 and T 6 are each independently (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m , 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines and esters; V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are each independently a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from SO2- and -P(O)OH-, and q is an integer of 1 to 6; During the ceremony, (PEG) nteeth [ka] where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: [ka] wherein y is an integer of 1 to 6, and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and AA is an amino acid residue, p is an integer from 1 to 20, and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring; Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0218] In certain embodiments, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 and V 6 is the following: is selected from During the ceremony, T 1 (C1~C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3 (PEG) n and V 3 is -CO-, T 4 is AA and V 4 does not exist, T 5 is PABC and V 5 does not exist, f is 0, or During the ceremony, T 1 (C1~C 12 ) alkyl, and V 1 is -CO-, T 2 is 4AP, and V 2 is -CO-, T 3 (C1~C 12 ) alkyl, and V 3 is -CO-, d, e and f are each 0.

[0219] For example, in certain embodiments, the conjugate of formula (I) is: [ka] The structure is selected from:

[0220] In certain embodiments, the left side of the linker structure is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety and the right side of the linker structure is attached to a drug W, e.g., as shown above. 1 is bonded to.

[0221] In certain embodiments, the conjugate is an antibody-drug conjugate, in which the antibody and drug are linked together by a linker (e.g., L), as described above. In some examples, the linker is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moieties include one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separable parts. For example, the cleavable moieties may include one or more covalent bonds that can dissociate or decompose under certain conditions to separate the cleavable linker into two or more parts. Thus, a cleavable linker can be included in an antibody-drug conjugate such that, under appropriate conditions, the cleavable linker is cleaved, separating or releasing the drug from the antibody at the drug's desired target site of action.

[0222] In some examples, the cleavable linker comprises two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of both cleavable moieties is required to separate or release the drug from the antibody at the desired target site of action of the drug. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that prevents cleavage of the first cleavable moiety. "Prevents cleavage" means that the presence of the uncleaved second cleavable moiety reduces or substantially inhibits the possibility of cleavage of the first cleavable moiety, thus substantially reducing the amount of cleavable linker or preventing cleavage. For example, the presence of the uncleaved second cleavable moiety can prevent cleavage of the first cleavable moiety. Inhibition of cleavage of the first cleavable moiety by the presence of the second cleavable moiety then substantially reduces or prevents release of the drug from the antibody, e.g., can substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action.

[0223] In some cases, the second cleavable moiety prevents the first cleavable moiety from cleaving, so cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the obstacle to cleavage of the first cleavable moiety, thus enabling cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety can cause the cleavable linker to dissociate or separate into two or more moieties as described above, releasing the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. Substantially means that about 10% or less cleavage of the first cleavable moiety occurs in the presence of an uncleavable second cleavable moiety, for example about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable moiety occurs in the presence of an uncleavable second cleavable moiety.

[0224] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thereby allowing cleavage of the first cleavable moiety, which results in cleavage of the cleavable linker, thereby separating or releasing the drug from the antibody at the drug's desired target site of action, as described above. In certain cases, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not result in cleavage of the cleavable linker (e.g., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0225] Each cleavable moiety included in the cleavable linker can be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety, and the second cleavable moiety can be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above by the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved by the enzymatic action of an enzyme, including, but not limited to, peptides, glycosides, etc. In some examples, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired target site of action, such as the desired target site of action of a drug released from an antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in significant amounts in other regions, such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety can be controlled so that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0226] For example, as described herein, antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, e.g., for the delivery of cancer therapeutics to a desired site of action where cancer cells are present. In some cases, enzymes such as the protease enzyme cathepsin B can be a biomarker for cancer that is overexpressed in cancer cells. The overexpression, and therefore localization, of certain enzymes in cancer can be used in conjunction with an enzymatically cleavable moiety included in the cleavable linker of an antibody-drug conjugate of the present disclosure to specifically release a drug at a desired site of action (e.g., the site (and overexpressed enzyme) of the cancer). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme that is overexpressed in cancer cells. For example, the enzyme can be the protease enzyme cathepsin B. Thus, in some examples, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by a protease enzyme such as cathepsin B.

[0227] In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide suitable for use in a cleavable linker and capable of being cleaved by the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as the enzymatically cleavable moiety include, for example, Val-Ala, Phe-Lys, etc. For example, the first cleavable moiety (e.g., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can comprise a peptide. The presence of an uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action of the drug. In some examples, one of the amino acid residues of the peptide comprising the first cleavable moiety is linked to or comprises a substituent, and the substituent comprises a second cleavable moiety. In some examples, the second cleavable moiety comprises a glycoside.

[0228] In some embodiments, the enzymatically cleavable moiety is a sugar moiety such as a glycoside (or glyosyl). In some cases, the glycoside can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside. The glycoside can be any glycoside or glycoside derivative suitable for use in a cleavable linker and that can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable moiety (e.g., a cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside. For example, in some embodiments, the first cleavable moiety comprises a peptide and the second cleavable moiety comprises a glycoside. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. In some examples, the second cleavable moiety is a glucuronide. In some instances, the second cleavable moiety is a galactoside. In some instances, the second cleavable moiety is a glucoside. In some instances, the second cleavable moiety is a mannoside. In some instances, the second cleavable moiety is a fucoside. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0229] A glycoside can be attached (e.g., covalently) to a cleavable linker via a glycosidic bond. The glycosidic bond can be linked to the cleavable linker via various types of bonds, including, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside can be cleaved from the cleavable linker to which it is attached by an enzyme (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). The glycoside can be removed or cleaved from the cleavable linker by any convenient enzyme capable of cleaving (hydrolyzing) the glycosidic bond connecting the glycoside to the cleavable linker. Examples of enzymes that can be used to mediate cleavage (hydrolysis) of the glycosidic bond linking the glycoside to the cleavable linker include glycosidases such as glucuronidase and galactosidase, glucosidases, mannosidases, fucosidases, and the like. Other suitable enzymes can also be used to mediate cleavage (hydrolysis) of the glycosidic bond linking the glycoside to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond linking the glycoside to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, e.g., a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where the enzyme mediating cleavage of the first cleavable moiety is found.

[0230] In certain embodiments, the conjugate of formula (I) is: [ka] The structure is selected from:

[0231] Any of the chemical entities, drugs, linkers and coupling moieties set forth in the descriptions and structures above can be adapted for use in the subject conjugates.

[0232] Further disclosure regarding methods for making hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl compounds and conjugates can be found in U.S. Patent Nos. 9,310,374 and 9,493,413, the disclosures of each of which are incorporated herein by reference. Further disclosure related to cleavable linkers can be found in U.S. Provisional Application No. 63 / 214,525, filed June 24, 2021, the disclosure of which is incorporated herein by reference.

[0233] An embodiment of the present disclosure is a compound of formula (II): [ka] wherein: Z 1 , Z 2 , Z 3 and Z 4 are each independently CR 24 , N and CL B -W 12 and at least one Z 1 , Z 2 , Z 3 and Z 4 is CL B -W 12 and R 21 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 22 and R 23are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 22 and R 23 may be cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 24 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker, L B is a second linker, W 11 is the first drug, W 12 is the second drug, W 13 is a polypeptide.

[0234] The substituents associated with the conjugates of formula (II) are described in more detail below.

[0235] In certain embodiments, Z 1 , Z 2 , Z 3 and Z 4 is CR 24 , N and CL B -W 12~ each independently selected and containing at least one Z 1 , Z 2 , Z 3 and Z 4 is CL B -W 12 In certain embodiments, Z 1 is CR 24 In certain embodiments, Z 1 is N. In certain embodiments, Z 1 is CL B -W 12 In certain embodiments, Z 2 is CR 24 In certain embodiments, Z 2 is N. In certain embodiments, Z 2 is CL B -W 12 In certain embodiments, Z 3 is CR 24 In certain embodiments, Z 3 is N. In certain embodiments, Z 3 is CL B -W 12 In certain embodiments, Z 4 is CR 24 In certain embodiments, Z 4 is N. In certain embodiments, Z 4 is CL B -W 12 is.

[0236] Various Z 1 , Z 2 , Z 3 and Z 4 For example, in some instances, Z 1 is CL B -W 12 and Z 2 is CR 24 and Z 3 is CR 24 and Z 4 is CR 24 In some cases, Z 1 is CR 24 and Z 2 is CLB -W 12 and Z 3 is CR 24 and Z 4 is CR 24 In some cases, Z 1 is CR 24 and Z 2 is CR 24 and Z 3 is CL B -W 12 and Z 4 is CR 24 In some cases, Z 1 is CR 24 and Z 2 is CR 24 and Z 3 is CR 24 and Z 4 is CL B -W 12 is.

[0237] In certain embodiments, R 21 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 21 is hydrogen. In certain embodiments, R 21 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 21 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl.21 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkynyl or substituted alkynyl, such as a substituted alkenyl. 21 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 21 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 21 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 21 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0238] In certain embodiments, R 22 and R 23are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 22 and R 23 may be cyclically linked to form a 5- or 6-membered heterocyclyl.

[0239] In certain embodiments, R 22 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 22 is hydrogen. In certain embodiments, R 22 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 22 is methyl. In certain embodiments, R 22 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 22 is alkynyl or substituted alkynyl. In certain embodiments, R 22 is alkoxy or substituted alkoxy. In certain embodiments, R 22 is amino or substituted amino. In certain embodiments, R 22 is carboxyl or carboxyl ester. In certain embodiments, R 22 is acyl or acyloxy. In certain embodiments, R 22 is acylamino or aminoacyl. In certain embodiments, R 22 is an alkylamide or a substituted alkylamide. In certain embodiments, R 22 is sulfonyl. In certain embodiments, R 22 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 22 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 22 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 22 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 22 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0240] In certain embodiments, R 23 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 23 is hydrogen. In certain embodiments, R 23 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 23 is methyl. In certain embodiments, R 23 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 23 is alkynyl or substituted alkynyl. In certain embodiments, R 23 is alkoxy or substituted alkoxy. In certain embodiments, R 23 is amino or substituted amino. In certain embodiments, R 23 is carboxyl or carboxyl ester. In certain embodiments, R 23 is acyl or acyloxy. In certain embodiments, R 23is acylamino or aminoacyl. In certain embodiments, R 23 is an alkylamide or a substituted alkylamide. In certain embodiments, R 23 is sulfonyl. In certain embodiments, R 23 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 23 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 23 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 23 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 23 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0241] In certain embodiments, R 22 and R 23 Both of the groups are methyl.

[0242] In certain embodiments, R 22 and R 23may be cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 22 and R 23 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl. 22 and R 23 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 22 and R 23 are cyclically linked to form a 6-membered heterocyclyl.

[0243] In certain embodiments, each R 24 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0244] Each R 24 The various possibilities for are described in more detail below. In certain embodiments, R 24 is hydrogen. In certain embodiments, each R 24 is hydrogen. In certain embodiments, R 24 is halogen, for example, F, Cl, Br, or I, and in certain embodiments, R 24 is F, and in certain embodiments, R 24 is Cl. In certain embodiments, R 24 is Br. In certain embodiments, R 24 is I. In certain embodiments, R 24 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 24 is methyl. In certain embodiments, R 24 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 24 is alkynyl or substituted alkynyl. In certain embodiments, R 24 is alkoxy or substituted alkoxy. In certain embodiments, R 24 is amino or substituted amino. In certain embodiments, R 24 is carboxyl or carboxyl ester. In certain embodiments, R 24 is acyl or acyloxy. In certain embodiments, R 24 is acylamino or aminoacyl. In certain embodiments, R 24 is an alkylamide or a substituted alkylamide. In certain embodiments, R 24 is sulfonyl. In certain embodiments, R 24 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 24 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl (e.g., phenyl or substituted phenyl). 24 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 24 is cycloalkyl or substituted cycloalkyl, e.g., C3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 24 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0245] In certain embodiments, L A is the first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0246] In certain embodiments, L B is the second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0247] In certain embodiments, W 11 is the first drug (or first active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0248] In certain embodiments, W 12 is a second drug (or second active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0249] In certain embodiments, W 13 is a polypeptide (e.g., an antibody). 13comprises one or more fGly' residues as described herein. In certain embodiments, the polypeptide is attached to the remainder of the conjugate via an fGly' residue as described herein. Examples of polypeptides and antibodies that can be used in the conjugates of the present disclosure are described in more detail below.

[0250] In certain embodiments, the conjugate of Formula (II) comprises a first linker, L A The first linker L A can be utilized to attach a first moiety of interest (e.g., a first drug or active agent) to a polypeptide (e.g., an antibody) via a conjugation moiety. A can be attached (e.g., covalently attached) to a conjugation moiety (e.g., as described herein). For example, the first linker L A can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a first drug. A hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety can be used to attach a first linker, L A The (and thus the first drug) can be conjugated to a polypeptide such as an antibody.

[0251] For example, as shown in the above formula (II), L A is conjugated to W via the conjugation moiety 13 Because it is bonded to W 13 is indirectly linked to the linker L via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety A As mentioned above, W 13 is a polypeptide (e.g., an antibody), so L A is attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, e.g., a linker L A is indirectly attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0252] Any convenient linker may be used in conjunction with the first linker L in the subject conjugates and compounds. A In certain embodiments, the first linker L A can comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker L A may comprise an alkyl or substituted alkyl group. In certain embodiments, the first linker L A may comprise an alkenyl or substituted alkenyl group. In certain embodiments, the first linker L A may comprise an alkynyl or substituted alkynyl group. In certain embodiments, the first linker, L A may comprise an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L A may comprise an amino group or a substituted amino group. In certain embodiments, the first linker L A may comprise a carboxyl group or a carboxyl ester group. In certain embodiments, the first linker L A may comprise an acylamino group. In certain embodiments, the first linker L A may comprise an alkylamide or substituted alkylamide group. In certain embodiments, the first linker L A may comprise an aryl or substituted aryl group. In certain embodiments, the first linker L A may comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L A may comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker L A may include a heterocyclyl or substituted heterocyclyl group.

[0253] In certain embodiments, the first linker L A can comprise a polymer. For example, the polymer can include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated in the conjugates and compounds described in more detail below.

[0254] In some embodiments, L A is the expression: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, wherein L is a first linker described by 1 , L 2 , L 3 , L 4 , L 5 and L 6 are each independently a linker subunit; and a, b, c, d, e, and f are each independently 0 or 1.

[0255] In certain embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 0. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0. In certain embodiments, a is 1, and b, c, d, e, and f are each 0.

[0256] In certain embodiments, the linker subunit L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 2 is the first drug or active agent W, if present 11 In certain embodiments, the linker subunit L 3 is the first drug or active agent W, if present 11 In certain embodiments, the linker subunit L 4 is the first drug or active agent W, if present 11 In certain embodiments, the linker subunit L 5 is the first drug or active agent W, if present 11 In certain embodiments, the linker subunit L 6 is the first drug or active agent W, if present 11is bonded to.

[0257] Any convenient linker subunit may be used as the first linker L A Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 and L 6 Each (when present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0258] In some embodiments, L 1 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 In some embodiments, L 1 comprises an aryl group or a substituted aryl group. In some embodiments, L 1 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0259] In some embodiments, L 2(when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 comprises an amino acid residue. In some embodiments, L 2 In some embodiments, L 2 comprises an aryl group or a substituted aryl group. In some embodiments, L 2 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0260] In some embodiments, L 3 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 comprises an amino acid residue. In some embodiments, L 3 In some embodiments, L 3 comprises an aryl group or a substituted aryl group. In some embodiments, L 3 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0261] In some embodiments, L 4 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 In some embodiments, L 4In some embodiments, L 4 comprises an amino acid residue. In some embodiments, L 4 In some embodiments, L 4 comprises an aryl group or a substituted aryl group. In some embodiments, L 4 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0262] In some embodiments, L 5 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L 5 comprises an amino acid residue. In some embodiments, L 5 In some embodiments, L 5 comprises an aryl group or a substituted aryl group. In some embodiments, L 5 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0263] In some embodiments, L 6 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 6 In some embodiments, L 6 In some embodiments, L 6 comprises an amino acid residue. In some embodiments, L 6 In some embodiments, L 6 comprises an aryl group or a substituted aryl group. In some embodiments, L6 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0264] In some embodiments, L A is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - a first linker comprising: -(L 1 ) a -is-(T 1 -V 1 ) a - and -(L 2 ) b -is-(T 2 -V 2 ) b - and -(L 3 ) c -is-(T 3 -V 3 ) c - and -(L 4 ) d -is-(T 4 -V 4 ) d - and -(L 5 ) e -is-(T 5 -V 5 ) e - and -(L 6 ) f -is-(T 6 -V 6 ) f - and In the formula, T 1 , T 2 , T 3 , T 4 , T 5 and T 6is a tether group, if present, V 1 , V 2 , V 3 , V 4 , V 5 and V 6 is a covalent bond or linking functional group, if present; a, b, c, d, e and f each independently represent 0 or 1.

[0265] In certain embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 0. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0. In certain embodiments, a is 1, and b, c, d, e, and f are each 0.

[0266] As noted above, in certain embodiments, L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (II) above). Thus, in certain embodiments, T 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (II) above). In certain embodiments, V 1is attached to the first drug or active agent. In certain embodiments, L 2 When present, T is attached to the first drug or active agent. 2 is attached to a first drug or active agent, if present, or V 2 When present, L is attached to the first drug or active agent. In certain embodiments, L 3 When present, T is attached to the first drug or active agent. 3 is attached to a first drug or active agent, if present, or V 3 When present, L is attached to the first drug or active agent. In certain embodiments, L 4 When present, T is attached to the first drug or active agent. 4 is attached to a first drug or active agent, if present, or V 4 When present, L is attached to the first drug or active agent. In certain embodiments, L 5 When present, T is attached to the first drug or active agent. 5 is attached to a first drug or active agent, if present, or V 5 When present, L is attached to the first drug or active agent. In certain embodiments, L 6 When present, T is attached to the first drug or active agent. 6 is attached to a first drug or active agent, if present, or V 6 When present, is attached to the first drug or active agent.

[0267] In certain embodiments, the conjugate of Formula (II) comprises a second linker, L B The second linker L B can be utilized to attach a second moiety of interest (e.g., a second drug or active agent) to the polypeptide (e.g., an antibody) via a conjugation moiety. Bcan be attached (e.g., covalently attached) to a conjugation moiety (e.g., as described herein). For example, a second linker, L B can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a second drug. A hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety can be used to attach a second linker, L B The (and thus the second drug) can be conjugated to a polypeptide such as an antibody.

[0268] For example, as shown in formula (II) above, L B via the conjugation moiety W 13 is bonded to W 13 is connected to a second linker L via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety B As mentioned above, W 13 is a polypeptide (e.g., an antibody), so L B is attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, e.g., a linker L B is indirectly attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0269] Any convenient linker may be used as the second linker, L, in the subject conjugates and compounds. B In certain embodiments, the second linker L Bcan comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker L B may comprise an alkyl or substituted alkyl group. In certain embodiments, the second linker, L B may comprise an alkenyl or substituted alkenyl group. In certain embodiments, the second linker, L B may comprise an alkynyl or substituted alkynyl group. In certain embodiments, the second linker, L B may comprise an alkoxy or substituted alkoxy group. In certain embodiments, the second linker, L B may comprise an amino group or a substituted amino group. In certain embodiments, the second linker, L B may comprise a carboxyl group or a carboxyl ester group. In certain embodiments, the second linker L B may comprise an acylamino group. In certain embodiments, the second linker, L B may comprise an alkylamide or substituted alkylamide group. In certain embodiments, the second linker, L B may comprise an aryl or substituted aryl group. In certain embodiments, the second linker, L B may comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker, L B may comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker, L B may include a heterocyclyl or substituted heterocyclyl group.

[0270] In certain embodiments, the second linker L Bcan comprise a polymer. For example, the polymer can include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated in the conjugates and compounds described in more detail below.

[0271] In some embodiments, L B is the expression: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m , wherein L is a second linker described by 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 are each independently a linker subunit; and g, h, i, j, k, l, and m are each independently 0 or 1.

[0272] In certain embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 0. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k, and l are each 1, and m is 0. In certain embodiments, g, h, i, j, and k are each 1, and l and m are each 0. In certain embodiments, g, h, i, and j are each 1, and k, l, and m are each 0. In certain embodiments, g, h, and i are each 1, and j, k, l, and m are each 0. In certain embodiments, g and h are each 1, and i, j, k, l, and m are each 0. In certain embodiments, g is 1, and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0.

[0273] In certain embodiments, the linker subunit L 7 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (II) above). In certain embodiments, the linker subunit L 8 If present, the second drug or active agent W 12 In certain embodiments, the linker subunit L 9 If present, the second drug or active agent W 12 In certain embodiments, the linker subunit L 10 If present, the second drug or active agent W 12In certain embodiments, the linker subunit L 11 If present, the second drug or active agent W 12 In certain embodiments, the linker subunit L 12 If present, the second drug or active agent W 12 In certain embodiments, the linker subunit L 13 If present, the second drug or active agent W 12 is bonded to.

[0274] Any convenient linker subunit may be used as the second linker L B Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 Each (when present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0275] In some embodiments, L 7 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 7 In some embodiments, L 7 In some embodiments, L 7comprises an amino acid residue. In some embodiments, L 7 In some embodiments, L 7 comprises an aryl group or a substituted aryl group. In some embodiments, L 7 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0276] In some embodiments, L 8 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 comprises an amino acid residue. In some embodiments, L 8 In some embodiments, L 8 comprises an aryl group or a substituted aryl group. In some embodiments, L 8 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0277] In some embodiments, L 9 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 comprises an amino acid residue. In some embodiments, L 9 In some embodiments, L 9 comprises an aryl group or a substituted aryl group. In some embodiments, L 9 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0278] In some embodiments, L 10 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 comprises an amino acid residue. In some embodiments, L 10 In some embodiments, L 10 comprises an aryl group or a substituted aryl group. In some embodiments, L 10 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0279] In some embodiments, L 11 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 comprises an amino acid residue. In some embodiments, L 11 In some embodiments, L 11 comprises an aryl group or a substituted aryl group. In some embodiments, L 11 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0280] In some embodiments, L 12 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 12In some embodiments, L 12 In some embodiments, L 12 comprises an amino acid residue. In some embodiments, L 12 In some embodiments, L 12 comprises an aryl group or a substituted aryl group. In some embodiments, L 12 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0281] In some embodiments, L 13 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 comprises an amino acid residue. In some embodiments, L 13 In some embodiments, L 13 comprises an aryl group or a substituted aryl group. In some embodiments, L 13 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0282] In some embodiments, L B is -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m a second linker comprising: -(L 7 ) g-is-(T 7 -V 7 ) g - and -(L 8 ) h -is-(T 8 -V 8 ) h - and -(L 9 ) i -is-(T 9 -V 9 ) i - and -(L 10 ) j -is-(T 10 -V 10 ) j - and -(L 11 ) k -is-(T 11 -V 11 ) k - and -(L 12 ) l -is-(T 12 -V 12 ) l - and -(L 13 ) m -is-(T 13 -V 13 ) m - and (In the formula, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 is a tether group, if present, V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 is a covalent bond or linking functional group, if present; g, h, i, j, k, l, and m each independently represent 0 or 1.

[0283] In certain embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 0. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k, and l are each 1, and m is 0. In certain embodiments, g, h, i, j, and k are each 1, and l and m are each 0. In certain embodiments, g, h, i, and j are each 1, and k, l, and m are each 0. In certain embodiments, g, h, and i are each 1, and j, k, l, and m are each 0. In certain embodiments, g and h are each 1, and i, j, k, l, and m are each 0. In certain embodiments, g is 1, and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0.

[0284] As noted above, in certain embodiments, L 7 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (II) above). Thus, in certain embodiments, T 7 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (II) above). In certain embodiments, V 7 is attached to a second drug or active agent. In certain embodiments, L 8 When present, T is attached to a second drug or active agent. 8is attached to a second drug or active agent, if present, or V 8 When present, L is attached to a second drug or active agent. In certain embodiments, L 9 When present, T is attached to a second drug or active agent. 9 is attached to a second drug or active agent, if present, or V 9 When present, L is attached to a second drug or active agent. In certain embodiments, L 10 When present, T is attached to a second drug or active agent. 10 is attached to a second drug or active agent, if present, or V10 4 When present, L is attached to a second drug or active agent. In certain embodiments, L 11 When present, T is attached to a second drug or active agent. 11 is attached to a second drug or active agent, if present, or V 11 When present, L is attached to a second drug or active agent. In certain embodiments, L 12 When present, T is attached to a second drug or active agent. 12 is attached to a second drug or active agent, if present, or V 12 When present, L is attached to a second drug or active agent. In certain embodiments, L 13 When present, T is attached to a second drug or active agent. 13 is attached to a second drug or active agent, if present, or V 13 When present, is attached to a second drug or active agent.

[0285] Tether group T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T8 , T 9 , T 10 , T 11 , T 12 and T 13 Any convenient tether group can be utilized in the subject linker. 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are covalent bonds, (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester, wherein each w is an integer of 1 to 20, each n is an integer of 1 to 30, each p is an integer of 1 to 20, and each x is an integer of 1 to 12.

[0286] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7, T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) is (C1~C 12 ) alkyl or substituted (C1-C 12 In certain embodiments, (C1-C 12 )Alkyl is a straight or branched alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, (C1-C 12 ) Alkyl is alkyl or substituted alkyl, for example C1-C 12 Alkyl or C1-C 10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) alkyl is C2-alkyl. For example, (C1-C 12 ) Alkyl is alkylene or substituted alkylene, for example C1-C 12 Alkylene or C1-C 10 It can be alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some cases, (C1-C 12 ) alkyl is C1-alkylene (e.g., CH2). In some cases, (C1-C 12 ) alkyl is C2-alkylene (e.g., CH2CH2). In some cases, (C1-C 12 ) Alkyl is C3-alkylene (e.g., CH2CH2CH2).

[0287] In certain embodiments, the substitution (C1-C 12 ) alkyl is a straight or branched substituted alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some examples, substituted (C1-C 12 ) Alkyl is a substituted C1-C12 Alkyl or substituted C1-C 10 It can be alkyl, or substituted alkyl, such as substituted C1-C6 alkyl, or substituted C1-C3 alkyl. In some cases, substituted (C1-C 12 ) alkyl is a substituted C2-alkyl. For example, substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkylene or substituted C-C 10 It can be alkylene, or substituted alkylene, such as substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some cases, the substituted (C1-C 12 ) alkyl is a substituted C1-alkylene (e.g., C1-alkylene substituted with —SO3H). In some cases, a substituted (C1-C 12 ) alkyl is a substituted C2-alkylene. In some cases, substituted (C1-C 12 ) alkyl is a substituted C3-alkylene. For example, substituted (C1-C 12 ) alkyl is (PEG) as defined herein k groups (e.g., -CONH(PEG) k , for example, -CONH(PEG)3 or -CONH(PEG)5; or -NHCO(PEG) k C1-C substituted with, for example, -NHCO(PEG) 12 It may contain alkylene (e.g., C3-alkylene or C5-alkylene) or C1-C substituted with -CONHCH2CH2SO3H groups. 12 It may contain alkylene (e.g., C3-alkylene) or C1-C substituted with -NHCOCH2SO3H groups. 12 It may include alkylene (eg, C5-alkylene).

[0288] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10, T 11 , T 12 and / or T 13 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some examples, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl is (C1-C 12 ) Alkyl, substituted (C1-C 12 ) may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some examples, the substituted aryl is a substituted phenyl and the substituent comprises a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0289] In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) includes heteroaryl or substituted heteroaryl, such as triazolyl (e.g., 1,2,3-triazolyl). In some cases, a tether group (e.g., T 1 , T 2 , T 3, T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) includes cycloalkyl or substituted cycloalkyl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) includes heterocyclyl or substituted heterocyclyl. In some examples, the substituent on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl includes a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0290] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) comprises an ethylenediamine (EDA) moiety, e.g., an EDA-containing tether group. In certain embodiments, (EDA) wcomprises one or more EDA moieties, e.g., where w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) moieties may be substituted at one or more convenient positions with any convenient substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety has the structure: [ka] , In the formula, y is an integer of 1 to 6, or 0 or 1, and each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be cyclically linked, for example to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 In certain embodiments, y is 1 and the adjacent R 12 The groups are selected from hydrogen, alkyl (eg, methyl), and substituted alkyl (eg, lower alkyl-OH, such as ethyl-OH or propyl-OH).

[0291] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be substituted at one or more convenient positions with any convenient substituent, such as an alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety has the structure: [ka] In the formula, R 12 is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxy-modified polyethylene glycol.

[0292] In certain embodiments, R 12 is a compound of the formula: (PEG) k which comprises a polyethylene glycol moiety described by the structure: [ka] , where k is an integer from 1 to 20, e.g., from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, k is 2. In certain embodiments, R 17 is selected from OH, COOH, OR, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 17 is COOH. In certain embodiments, R 17 is OH. In certain embodiments, R 17 is OCH3.

[0293] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) is (PEG) n (PEG) n is a polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG) n The structure: [ka] , In the formula, n is an integer from 1 to 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0294] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) is (AA) p where AA is an amino acid residue. Any convenient amino acid can be utilized. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary α-amino acids and β-alanine, and non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids. In certain embodiments, p is an integer between 1 and 50, e.g., between 1 and 40, 1 and 30, 1 and 20, 1 and 12, or 1 and 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0295] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T13 ) include amino acid analogs. Amino acid analogs include compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some examples, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, with the difference(s) being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl) or atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (such as a single bond, such as a double bond), or a combination thereof. For example, amino acid analogs may include alpha-hydroxy acids and alpha-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfoalanine, etc.

[0296] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) is the formula -(CR 13 OH) x-, where x is 0 or x is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 13 is hydrogen. In certain embodiments, R 13 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 13 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. In certain embodiments, R 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 13 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 13 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 13 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 13 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0297] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are selected from R13 is as described above.

[0298] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises a hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

[0299] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 ) includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP) or para-hydroxy-phenyl (PHP).

[0300] In some embodiments, the tether group has the following structure: [ka] The MABO group is described by:

[0301] In some embodiments, the tether group has the following structure: [ka] This includes the MABC group described by:

[0302] In some embodiments, the tether group has the following structure: [ka] This includes the PABO group described by:

[0303] In some embodiments, the tether group has the following structure: [ka] This includes the PABC group described by:

[0304] In some embodiments, the tether group has the following structure: [ka] The PAB group includes the PAB group described by:

[0305] In some embodiments, the tether group has the following structure: [ka] The PABA group is described by:

[0306] In some embodiments, the tether group has the following structure: [ka] This includes the PAP group described by:

[0307] In some embodiments, the tether group has the following structure: [ka] The PHP group includes the PHP group described by:

[0308] In certain embodiments, each R 14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0309] In certain embodiments, R 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R 14 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 14 is alkynyl or substituted alkynyl. In certain embodiments, R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14is acyl or acyloxy. In certain embodiments, R 14 is acylamino or aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 14 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 14 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 14 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 14 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0310] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more further groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0311] In certain embodiments, the tether group T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and / or T 13 Each of one or more of T may be substituted with a glycoside or glycoside derivative. For example, in some instances, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 Each of the groups may be substituted with a glycoside. 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 may each be substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0312] In certain embodiments, the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0313] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] may be selected from:

[0314] Linking functional group, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 With respect to V, any convenient linking functional group may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoridate, and the like. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V10 , V 11 , V 12 and V 13 is a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 is independently selected from SO2- and -P(O)OH-, and q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0315] In some embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0316] In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is alkyl or substituted alkyl, e.g., C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3Alkyl or C 1~3 In certain embodiments, R 15 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 15 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R 15 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, such as a C heteroaryl or a C substituted heteroaryl. 15 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R 15 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 It is a substituted heterocyclyl.

[0317] In certain embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are independently selected from R 15 is as described above.

[0318] As noted above, in some embodiments, L A is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) fwherein a, b, c, d, e, and f are each independently 0 or 1.

[0319] In some embodiments, the first linker L A In: T 1 is (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 and T 6 are each independently (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x , 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines and esters; V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are each independently a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from SO2- and -P(O)OH-, and q is an integer of 1 to 6; During the ceremony, (PEG) n teeth [ka] where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: [ka] wherein y is an integer of 1 to 6, and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and AA is an amino acid residue, p is an integer from 1 to 20, and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring; Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0320] In certain embodiments, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 and V 6 is the following: is selected from: T 1 (C1~C 12 ) alkyl, and V 1 is -CONH-.

[0321] T 2 is a substitution (C1~C 12 ) alkyl, and V 2 is -CO-); T 3 is AA and V 3 does not exist; T 4 is PABC and V 4 does not exist; and e and f are each 0.

[0322] In certain embodiments, the first linker L A The left side of the above linker structure for is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and a first linker L A The right side of the above linker structure for is attached to a first drug or active agent.

[0323] As described above, in some embodiments, L B is -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) m wherein g, h, i, j, k, l, and m are each independently 0 or 1.

[0324] In some embodiments, the second linker L B In: T 7 is (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x , 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines and esters; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 are each independently a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from SO2- and -P(O)OH-, and q is an integer of 1 to 6; During the ceremony, (PEG) n teeth [ka] where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: [ka] wherein y is an integer of 1 to 6, and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and AA is an amino acid residue, p is an integer from 1 to 20, and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring; Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0325] Any convenient tether group can be used as T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 For example, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 Any of the tether groups described above with respect to the tether group T 7, T 8 , T 9 , T 10 , T 11 , T 12 and T 13 can be used for.

[0326] V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 Any convenient linking functional group can be utilized for V. 1 , V 2 , V 3 , V 4 , V 5 and V 6 Any of the linking functional groups described above with respect to the linking functional group V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 can be used for.

[0327] In certain embodiments, each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are independently selected from R 13 is as described above.

[0328] In certain embodiments, each R 15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are independently selected from R 15 In these embodiments, the various possible substituents are as described above for R 15 is as described above.

[0329] Second linker L B In certain embodiments, the tether group T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 may be substituted with a glycoside or glycoside derivative, respectively. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0330] Second linker L BIn certain embodiments, the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the above MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0331] In certain embodiments, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 is the following: is selected from During the ceremony, T 7 does not exist, and V 7 is -NHCO-, T 8 is (C1~C 12 ) alkyl, and V 8 is -CONH-, T 9 is a substitution (C1~C 12 ) alkyl, and V 9 is -CO-, T 10 is AA and V 10 does not exist, T 11 is PABC and V 11 does not exist, l and m are each 0.

[0332] In certain embodiments, the second linker L B The left side of the above linker structure is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and a second linker L B The right side of the above linker structure is attached to a second drug or active agent.

[0333] In certain embodiments, the conjugate is an antibody-drug conjugate in which the antibody and the drug are linked to each other by a linker as described above. In some cases, the linker m (e.g., L A and / or L B ) is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moieties include one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separable parts. For example, the cleavable moiety may include one or more covalent bonds, which can dissociate or decompose under certain conditions to separate the cleavable linker into two or more parts. Thus, the linker included in the antibody-drug conjugate can be a cleavable linker such that the cleavable linker is cleaved under appropriate conditions, separating or releasing the drug from the antibody at the drug's desired target site of action.

[0334] In some examples, the cleavable linker comprises two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of both cleavable moieties is required to separate or release the drug from the antibody at the desired target site of action of the drug. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that prevents cleavage of the first cleavable moiety. "Prevents cleavage" means that the presence of the uncleaved second cleavable moiety reduces or substantially inhibits the possibility of cleavage of the first cleavable moiety, thus substantially reducing the amount of cleavable linker or preventing cleavage. For example, the presence of the uncleaved second cleavable moiety can prevent cleavage of the first cleavable moiety. Inhibition of cleavage of the first cleavable moiety by the presence of the second cleavable moiety then substantially reduces or prevents release of the drug from the antibody, e.g., can substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action.

[0335] In some cases, the second cleavable moiety prevents the first cleavable moiety from cleaving, so cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the obstacle to cleavage of the first cleavable moiety, thus enabling cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety can cause the cleavable linker to dissociate or separate into two or more moieties as described above, releasing the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. Substantially means that about 10% or less cleavage of the first cleavable moiety occurs in the presence of an uncleavable second cleavable moiety, for example about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable moiety occurs in the presence of an uncleavable second cleavable moiety.

[0336] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thereby enabling cleavage of the first cleavable moiety, which results in cleavage of the cleavable linker, thereby separating or releasing the drug from the antibody at the drug's desired target site of action, as described above. In certain cases, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0337] Each cleavable moiety included in the cleavable linker can be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety, and the second cleavable moiety can be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above by the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved by the enzymatic action of an enzyme, including, but not limited to, an ester, a peptide, or a glycoside. In some examples, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired target site of action, such as the desired target site of action of a drug released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in significant amounts in other regions, such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety can be controlled so that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0338] For example, as described herein, the antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, e.g., for the delivery of cancer therapeutics to a desired site of action where cancer cells are present. In some cases, enzymes such as esterases, which cleave ester bonds, or glycosidases, which cleave glycosidic bonds, can be biomarkers of cancer that are overexpressed in cancer cells. The overexpression, and therefore localization, of specific enzymes in cancer can be used in conjunction with an enzymatically cleavable moiety included in the cleavable linker of an antibody-drug conjugate of the present disclosure to specifically release a drug at a desired site of action (i.e., the site (and overexpressed enzyme) of the cancer). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or glycoside) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be an esterase. Thus, in some examples, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme. In some examples, the enzyme can be a glycosidase. Thus, in some instances, the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme.

[0339] In certain embodiments, the enzymatically cleavable moiety is an ester bond. For example, the first cleavable moiety (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can comprise an ester. The presence of an uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by an esterase enzyme, thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action. In some examples, a portion of the linker adjacent to the first cleavable moiety is linked to or comprises a substituent, and the substituent comprises a second cleavable moiety. In some examples, the second cleavable moiety comprises a glycoside or a glycoside derivative.

[0340] In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycosyl) or glycoside derivative. In some cases, the glycoside or glycoside derivative can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside or glycoside derivative. The glycoside or glycoside derivative can be any glycoside or glycoside derivative suitable for use in a cleavable linker that can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable moiety comprises an ester and the second cleavable moiety comprises a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some examples, the second cleavable moiety is a glucuronide. In some examples, the second cleavable moiety is a galactoside. In some examples, the second cleavable moiety is a glucoside. In some examples, the second cleavable moiety is a mannoside. In some examples, the second cleavable moiety is a fucoside. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0341] The glycoside or glycoside derivative can be covalently attached to the cleavable linker via a glycosidic bond. The glycosidic bond can be attached to the cleavable linker via various types of bonds, including, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside or glycoside derivative can be cleaved from the cleavable linker to which it is attached by an enzyme (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). The glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme capable of cleaving (hydrolyzing) the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. Examples of enzymes that can be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker are glycosidases, such as glucuronidase, galactosidase, glucosidase, mannosidase, and fucosidase. Other suitable enzymes can also be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, e.g., a lysosomal glycosidase, found in cells at or near the desired site of action of the drug of the antibody-drug conjugate. Optionally, the enzyme is an enzyme found at or near the target site where the enzyme that mediates cleavage of the first cleavable moiety is found.

[0342] Examples of conjugates according to the present disclosure include, but are not limited to, those having the following structure: [ka] Examples include:

[0343] Any of the chemical entities, linkers and conjugation moieties set forth in the structures above can be adapted for use in the subject compounds and conjugates.

[0344] Further disclosure regarding methods for preparing hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl compounds and conjugates can be found in U.S. Pat. Nos. 9,310,374 and 9,493,413, the disclosures of each of which are incorporated herein by reference.

[0345] Anti-MUC1 antibody As noted above, the subject conjugates include a substituent W 2 wherein the amino acid sequence of the anti-MUC1 antibody has been modified to include 2-formylglycine (fGly) residues. As used herein, amino acids may be referred to by standard names, standard three-letter abbreviations, and / or standard one-letter abbreviations, such as alanine or Ala or A; cysteine ​​or Cys or C; aspartic acid or Asp or D; glutamic acid or Glu or E; phenylalanine or Phe or F; glycine or Gly or G; histidine or His or H; isoleucine or Ile or I; lysine or Lys or K; leucine or Leu or L; methionine or Met or M; asparagine or Asn or N; proline or Pro or P; glutamine or Gln or Q; arginine or Arg or R; serine or Ser or S; threonine or Thr or T; valine or Val or V; tryptophan or Trp or W; and tyrosine or Tyr or Y.

[0346] According to some embodiments, the antibodies of the present disclosure specifically bind to MUC1 and include:

[0347] A variable heavy (VH) chain comprising heavy chain CDR1-3 (HCDR1-3) of the VH chain having the following sequence: EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRPGKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSSLRSEDTAVYYCARGLRYALDYWGQGTLVTVSS (SEQ ID NO: 1); and A variable light (VL) chain comprising light chain CDR1-3 (LCDR1-3) of the VL chain having the following sequence: EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQAPRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYAWSPPTFGQGTKLEIK (SEQ ID NO: 2); EIVLTQSPATLSLSPGERATLSCRASSSVGSSNLYWYQQKPGQAPRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYRWSPPTFGQGTKLEIK (SEQ ID NO: 3); or EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQAPRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYSWSPPTFGQGTKLEIK (SEQ ID NO: 4). HCDR1-3 and LCDR1-3 may be as defined by Chothia, Kabat, or IMT nomenclature. HCDR1-3 of the anti-MUC1 antibodies disclosed herein defined according to the recited nomenclature may be as follows: [Table 2] LCDRs 1-3 of the anti-MUC1 antibodies disclosed herein may be defined according to the nomenclature listed in Tables 3-5. [Table 3] [Table 4] [Table 5] In certain embodiments, the VH chain of the anti-MUC1 antibody comprises HCDR1-3 as described herein, and the VL chain of the anti-MUC1 antibody comprises LCDR1-3, wherein: LCDR1 comprises the amino acid sequence RASSSVG / SSSYLY (SEQ ID NO: 41); LCDR2 comprises the amino acid sequence G / RT / SS / TN / KLAS (SEQ ID NO: 42); LCDR3 contains the amino acid sequence HQYA / R / SWSPPT (SEQ ID NO: 43) as defined by Kabat.

[0348] In certain embodiments, the VH chain of an anti-MUC1 antibody comprises HCDRs 1-3 as described herein and comprises an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, any amino acid differences between the VH chain of an anti-MUC1 antibody of the disclosure and SEQ ID NO: 1 may be limited to regions outside of the CDRs, e.g., one or more of the framework regions (FRs), such as FR1, FR2, FR3, and / or FR4.

[0349] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR1-3 as set forth in Table 3 herein, and comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0350] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR1-3 as set forth in Table 4 herein, and comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.

[0351] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR1-3 as set forth in Table 5 herein, and comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0352] In certain embodiments, any amino acid differences between the VL chain of an anti-MUC1 antibody of the disclosure and SEQ ID NOs: 2, 3, and 4 may be limited to regions outside of the CDRs, e.g., one or more of the framework regions (FRs), such as FR1, FR2, FR3, and / or FR4.

[0353] In certain embodiments, an anti-MUC1 antibody of the disclosure may comprise: a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1; and b) a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO: 2, 3, or 4.

[0354] In certain embodiments, an anti-MUC1 antibody of the present disclosure may comprise: a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the amino acid sequence set forth in any one of SEQ ID NOs: 57-73; and b) a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO: 2, 3, or 4.

[0355] In certain embodiments, an anti-MUC1 antibody of the present disclosure may comprise: a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the amino acid sequence set forth in any one of SEQ ID NOs: 57-73, wherein C in the constant region sequence LCTPSR is replaced with fGly; and b) a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO: 2, 3, or 4.

[0356] In certain embodiments, an anti-MUC1 antibody of the present disclosure may comprise: a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the amino acid sequence set forth in any one of SEQ ID NOs: 57-73, wherein C present in the constant region sequence LCTPSR is replaced with fGly', where fGly' represents the amino acid residue conjugated to a moiety of interest; and b) a light chain comprising a VL region having the amino acid sequence set forth in SEQ ID NO: 2, 3, or 4.

[0357] In certain embodiments, an anti-MUC1 antibody of the present disclosure may comprise: a) a heavy chain comprising a VH region comprising an amino acid sequence at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 1, and a heavy chain constant region comprising an amino acid sequence at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in any one of SEQ ID NOs: 57-73, wherein a C present in the sequence LCTPSR in the constant region is replaced by fGly', where fGly' refers to an amino acid residue conjugated to a moiety of interest; and b) a light chain comprising a VL region comprising an amino acid sequence at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 2, 3, or 4.

[0358] The anti-MUC1 antibodies of the present disclosure may bind to MUC-1 with an EC50 of about 0.4-1 nM, e.g., 0.5-0.9 nM, 0.6-0.8 nM, or 0.65-0.75 nM, as determined by ELISA. The EC50 is determined as the concentration of antibody providing a half-maximal response (e.g., half of the maximum fluorescence intensity). The MUC-1 may be a 20-mer glycosylated MUC1 peptide as disclosed in Example 1.

[0359] The anti-MUC1 antibodies of the present disclosure, as disclosed in Example 1, can bind to a 20-mer MUC1 glycosylated peptide, but not to a recombinant 60-mer MUC1 aglycosylated peptide.

[0360] The anti-MUC1 antibodies of the disclosure may bind to cancerous tissue and may not exhibit binding to normal tissue (e.g., little binding as measured by immunohistochemistry or undetectable binding by immunohistochemistry). For example, the anti-MUC1 antibodies described herein may bind to human stomach, breast, and / or lung tissue that has cancerous cells, but not exhibit detectable binding to human stomach, breast, and / or lung tissue that does not have cancerous cells.

[0361] The antibodies may be used in a variety of research, diagnostic, and therapeutic applications, including for carrying out any of the methods described in U.S. Patent Application Nos. 20120141375 and 20160145343, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0362] The subject antibodies specifically bind to a MUC1 polypeptide, wherein the epitope comprises amino acid residues within the human MUC1 antigen, which comprises the amino acid sequence set forth in SEQ ID NO:20: In certain embodiments, the MUC1 epitope bound by the anti-MUC1 antibodies disclosed herein is glycosylated. In certain embodiments, the MUC1 epitope bound by the anti-MUC1 antibodies disclosed herein is present on MUC1 expressed by upper lung adenocarcinoma cell lines and lung epithelial cells.

[0363] The subject antibodies exhibit high affinity binding to MUC1. For example, the subject antibodies exhibit high affinity binding to MUC1. -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M or 10 -12 The antibody binds to MUC1 at a binding affinity of greater than 10 -7 M ~ about 10 -8 M, about 10 -8 M ~ about 10 -9 M, about 10 -9 M ~ about 10 -10 M, about 10 -10 M ~ about 10 -11 M, or about 10 -11 M ~ about 10 -12 M, 10 -12 It binds to an epitope present on MUC1 with an affinity greater than M.

[0364] Anti-MUC1 antibodies of the disclosure can, in some cases, induce apoptosis in cells that express MUC1 on their cell surface.

[0365] A "MUC1 antigen" or "MUC1 polypeptide" can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to SEQ ID NO:20.

[0366] As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains (approximately 25 kD or 214 amino acids) are encoded by a variable region gene (approximately 110 amino acids) at the N-terminus and a kappa or lambda constant region at the C-terminus. Full-length immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) are encoded by a variable region gene (approximately 116 amino acids) at the N-terminus and one of the other aforementioned constant region genes, e.g., gamma (encoding approximately 330 amino acids), at the C-terminus. In some embodiments, the subject antibodies comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0367] In some embodiments, the subject antibodies do not comprise full-length immunoglobulin heavy chains and full-length immunoglobulin light chains, but instead comprise antigen-binding fragments of full-length immunoglobulin heavy chains and full-length immunoglobulin light chains. In some embodiments, the antigen-binding fragments are comprised on separate polypeptide chains. In other embodiments, the antigen-binding fragments are comprised within a single polypeptide chain. The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that are capable of specifically binding to MUC1, as described above. Examples of binding fragments include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment (consisting of the VH and CH1 domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; and (vii) a single-chain Fv (scFv) (a fragment synthesized using recombinant means such that the VH and VL domains pair to form a monovalent molecule). (viii) diabodies (consisting of two scFvs linked such that the VH and VL domains do not pair to form a monovalent molecule; the VH of each scFv pairs with the VL domain of the other scFv to form a divalent molecule); and (ix) bispecific antibodies (consisting of at least two antigen-binding regions, each region binding to a different epitope). In some embodiments, the antibody fragment of interest is a Fab fragment. In some embodiments, the antibody fragment of interest is a single-chain antibody (scFv).

[0368] In some embodiments, the subject antibody is a recombinant or engineered antibody, such as a chimeric antibody, a humanized antibody, a deimmunized antibody, or an in vitro-generated antibody. As used herein, the term "recombinant" or "engineered" antibody is intended to include all antibodies prepared, expressed, generated, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant combinatorial antibody library; (iii) antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes; or (iv) antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies include humanized antibodies, CDR-grafted antibodies, chimeric antibodies, deimmunized antibodies, and in vitro-generated antibodies. They may contain constant regions derived from human germline immunoglobulin sequences.

[0369] Full-length bispecific antibodies can be generated, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions into the heavy chain CH3 interface of each half molecule to promote heterodimerization of two antibody half molecules with different specificities, either in vitro in a cell-free environment or coexpression. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation / association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation / association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding to a different epitope.

[0370] Full-length bispecific antibodies can be generated using the "knob-in-hole" strategy (e.g., PCT Publication No. WO 2006 / 028936). Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After coexpression of the two antibodies, heterodimers are formed as a result of preferential interaction of the heavy chain with the "knob" and the heavy chain with the "hole." Exemplary CH3 substitution pairs that form knobs and holes are (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S / L368A / Y407V.

[0371] Other strategies may also be used, such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in published U.S. Patent Application Publication No. 2010 / 0015133; U.S. Patent Application Publication No. 2009 / 0182127; U.S. Patent Application Publication No. U.S. Pat. No. 82010 / 028637, or U.S. Patent Application Publication No. U.S. Pat. No. 2011 / 0123532. In another strategy, as described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849, heterodimerization can be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351Y / F405A / Y407V / T394W, T366 I / K392M / T394W / F405A / Y407V, T366L / K392M / T394W / F405A / Y407V, L351Y / Y407A / T366A / K409F, L351Y / Y407A / T366V / K409F, Y407A / T366A / K409F, or T350V / L351Y / F405A / Y407V, T350V / T366L / K392L / T394W.

[0372] Single-chain bispecific antibodies are also provided. In some embodiments, the single-chain bispecific antibodies of the present disclosure are bispecific scFvs. The subject antibodies may be humanized. The constant region(s), if present, may also be substantially or entirely derived from a human immunoglobulin.

[0373] Methods for producing humanized antibodies are known in the art. Substitution of mouse CDRs into a human variable domain framework can result in the retention of their correct spatial orientation, for example, if the human variable domain framework adopts the same or similar conformation as the mouse variable framework from which the CDRs are derived. This can be achieved by obtaining the human variable domain from a human antibody whose framework sequences exhibit a high degree of sequence identity with the mouse variable framework domain from which the CDRs are derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies or can be consensus sequences of several human antibodies.

[0374] Having identified the complementarity-determining regions of the murine donor immunoglobulin and a suitable human acceptor immunoglobulin, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with murine residues should be minimized, as the introduction of murine residues increases the risk that the antibody will elicit a human-anti-mouse-antibody (HAMA) response in humans. Art-recognized methods for determining immune responses can be performed to monitor HAMA responses in specific patients or during clinical trials. Patients receiving a humanized antibody can be given an immunogenicity assessment at the start of treatment and throughout administration. HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0375] Specific amino acids from the human variable region framework residues are selected for substitution based on their potential effect on CDR conformation and / or binding to antigen. The non-natural juxtaposition of the mouse CDR region and the human variable framework region may result in non-natural conformational constraints that result in loss of binding affinity unless corrected by substitution of certain amino acid residues. The selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are generated starting from the solved structure of an immunoglobulin chain or its domain. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the solved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for construction of the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably chains or domains sharing at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain being modeled and the amino acids in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0376] When framework residues as defined by Kabat, supra, constitute structural loop residues as defined by Chothia, supra, amino acids present in a murine antibody can be selected for substitution into a humanized antibody. Residues "adjacent to the CDR region" include amino acid residues located immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, such as the CDRs defined by Kabat or the CDRs defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids interact with amino acids in the CDRs and, if selected from the acceptor, are particularly likely to distort the donor CDR and reduce affinity. Furthermore, adjacent amino acids can directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to maintain all of the antigen contacts that provide affinity in the original antibody.

[0377] In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibodies are scFv dimers (e.g., comprising two tandem scFvs (scFv2)), scFv trimers (e.g., comprising three tandem scFvs (scFv3)), scFv tetramers (e.g., comprising four tandem scFvs (scFv4)), or multimers of more than four scFvs (e.g., in tandem). The scFv monomers can be linked in tandem via a linker about 2 to about 10 amino acids in length, e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkers include, for example, (Gly) x (wherein x is an integer of 2 to 10), glycine-serine polymers, etc.

[0378] In certain embodiments, the antibody is conjugated to the drug via a cleavable or non-cleavable linker. Linkers suitable for use with the subject antibodies include "flexible linkers." When present, the linker molecule is generally long enough to allow some flexible movement between the linked regions. Linker molecules are generally about 6 to 50 atoms in length. Linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides may be used in light of the present disclosure.

[0379] According to some embodiments, the linker is a chemically labile linker, e.g., an acid-cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into target cells (e.g., cancer cells) by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of the target cells. Enzyme-labile linkers include, but are not limited to, linkers containing a peptide bond, e.g., dipeptide-based linkers, such as a valine-citrulline (VC) linker, e.g., a maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, a valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, and the like.

[0380] In some embodiments, a subject antibody comprises an immunoglobulin constant region (e.g., an Fc region). The Fc region, if present, can be a human Fc region. If a constant region is present, the antibody can comprise both a light chain constant region and a heavy chain constant region. The antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is the human isotype IgG1Fc. The light chain constant region can be lambda or kappa. A subject antibody (e.g., a subject humanized antibody) can comprise sequences from more than one class or isotype. An antibody can be expressed as a tetramer comprising two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab'), and Fv, or as a single-chain antibody in which the heavy and light chain variable domains are linked via a spacer.

[0381] In some embodiments, anti-MUC1 antibodies of the disclosure may include one or more amino acid substitutions introduced into the Fc region. In some embodiments, one or more of the amino acid substitutions may be at positions 239, 298, 326, 330, and 332 in the Fc region. In some embodiments, anti-MUC1 antibodies of the disclosure may include one or more of the following amino acid substitutions introduced into the Fc region: I332E; S239D / A330L / I332E; S239D / S298A / I332E; S239D / K326T / I332E; S239D / S298A / K326T / I332E; or S239D / A330L / I332E / D356E / L358M.

[0382] In some embodiments, the subject antibodies comprise a free thiol (-SH) group at the carboxyl terminus, which can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, etc.

[0383] In some embodiments, the subject antibodies comprise one or more unnatural amino acids. In some embodiments, the non-naturally encoded amino acids comprise a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. The inclusion of non-naturally occurring amino acids can provide for attachment to a polymer, a second polypeptide, a scaffold, or the like. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0384] The present disclosure also provides anti-MUC1 antibodies bearing a binding moiety of interest, e.g., a detectable label, a drug, a half-life extending moiety, etc. Modification of the antibody can be achieved by a variety of synthetic and / or recombinant methods. The one or more moieties attached to the antibody can provide one or more of a wide variety of functions or characteristics. Exemplary moieties include detectable labels (e.g., dye labels (e.g., chromophores, fluorophores), biophysical probes (spin labels, nuclear magnetic resonance (NMR) probes), fluorescence resonance energy transfer (FRET) type labels (e.g., at least one member of a FRET pair comprising at least one member of a fluorophore / quencher pair), bioluminescence resonance energy transfer (BRET) type labels (e.g., at least one member of a BRET pair), immunodetectable tags (e.g., FLAG, His(6), etc.), water-soluble polymers (e.g., PEGylation); purification tags (e.g., to facilitate isolation by affinity chromatography (e.g., attachment of a FLAG epitope); membrane localization domains (e.g., lipid or carbohydrate phosphatidylinositol (GPI)-type anchors); immobilization tags (e.g., to facilitate attachment of a polypeptide to a surface, including selective attachment); drugs (e.g., to facilitate drug targeting, e.g., via conjugation of a drug to an antibody); and the like.

[0385] In some embodiments, a subject antibody is linked (e.g., covalently attached) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally occurring polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene, polyalkenylene, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.Suitable polymers include, for example, ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoesters; polyanhydrides; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonat poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-esters) (e.g., poly(ethylene oxide)-poly(lactic acid) (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and Examples of suitable polymers include ethylene-alpha olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyvinyl aromatics such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers and olefins, such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; polyamides such as nylon 66 and polycaprolactam; alkyd resins; polycarbonate; polyoxymethylene; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon®; poly(ethylene glycol); and carboxymethyl cellulose.

[0386] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and derivatives thereof, e.g., substituted poly(ethylene glycol), such as methoxypoly(ethylene glycol), and derivatives thereof. Suitable naturally occurring polymers include, e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.

[0387] Suitable polymers can have an average molecular weight ranging from 500 Da to 50,000 Da, e.g., from 5,000 Da to 40,000 Da, or from 25,000 to 40,000 Da. For example, in some embodiments, when a subject antibody comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer can have a molecular weight ranging from about 0.5 kilodaltons (kDa) to 1 kDa, from about 1 kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25 kDa, from 25 kDa to 40 kDa, or from 40 kDa to 60 kDa.

[0388] In some embodiments, a subject antibody is covalently attached to a PEG polymer. In some embodiments, a subject scFv multimer is covalently attached to a PEG polymer. PEGs suitable for conjugation to proteins are generally soluble in water at room temperature and have the general formula R(O-CH-CH). n The PEG has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has between 1 and 8 carbons. The PEG conjugated to the subject antibody can be linear or branched. Branched PEG derivatives include star PEG and multi-arm PEG.

[0389] A subject antibody may be glycosylated, e.g., the subject antibody may comprise a covalently bound carbohydrate or polysaccharide moiety. Glycosylation of antibodies is typically either N-linked or O-linked. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence such that it contains an N- or O-linked glycosylation site. Similarly, removal of glycosylation sites can be accomplished by altering amino acids within the antibody's native glycosylation sites.

[0390] The antibody of interest can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the antibody of interest, a synthetic polymer, a carbohydrate, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides through their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At mildly alkaline pH, imidoesters react only with primary amines to form imidoamides, leaving the overall charge of the crosslinked polypeptides unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propinoamidobutane (DPDPB).

[0391] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety) that crosslink with one polypeptide via the amine- or sulfhydryl-reactive moiety and then react with the other polypeptide via the unreacted moiety. Similar to pyridyl disulfide crosslinkers, several heterobifunctional haloacetyl crosslinkers are available. Carbodiimides are a classic example of heterobifunctional crosslinking reagents for coupling carboxyls to amines, resulting in amide bonds.

[0392] The subject antibodies, in some embodiments, comprise a "radiopaque" label, e.g., a label that can be readily visualized using, for example, X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide salts, bromide salts, or barium salts. Other radiopaque materials are also known, including, but not limited to, organobismuth derivatives, radiopaque polyurethanes, organobismuth complexes, radiopaque barium multimer complexes, and the like.

[0393] In some embodiments, the subject antibodies comprise a polyamine modification. The subject antibodies can be modified with either naturally occurring or synthetic polyamines. Useful natural polyamines include putrescine, spermidine, spermine, 1,3-deaminopropane, norspermidine, syn-homospermidine, thermon, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines can be represented by the empirical formula C X H Y N Z and may be a cyclic or acyclic, branched or unbranched hydrocarbon chain of 3 to 12 carbon atoms consisting of, and further containing 1 to 6 NR or N(R)2 moieties, where R is H, (C1-C4) alkyl, phenyl, or benzyl. The polyamine can be linked to the antibody using any standard cross-linking method.

[0394] When an anti-MUC1 antibody of the disclosure includes a covalently attached heterologous moiety, the heterologous moiety can be linked to the anti-MUC1 heavy and / or light chain directly or via a linker. Suitable linkers can be readily selected and can be any of a variety of lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, e.g., from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0395] Examples of flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 21) and (GGGS) n (SEQ ID NO: 22), where n is an integer of at least 1), including glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0396] Antibody modification methods An anti-MUC1 antibody conjugate of the present disclosure can comprise: 1) an Ig heavy chain constant region conjugated to a moiety of interest; and an Ig light chain constant region conjugated to a moiety of interest; 2) an Ig heavy chain constant region conjugated to a moiety of interest; and an Ig light chain constant region not conjugated to a moiety of interest; or 3) an Ig heavy chain constant region not conjugated to a moiety of interest; and an Ig light chain constant region conjugated to a moiety of interest. The subject anti-MUC1 antibody conjugates can also comprise a VH and / or VL domain conjugated to a moiety of interest.

[0397] In one example, an antibody can be modified to contain a 2-formylglycine residue, which can serve as a chemical handle for attachment of a heterologous moiety. For example, the heavy and / or light chain constant regions of an anti-MUC1 antibody of the present disclosure can be modified to contain the amino acid sequence of a sulfatase motif that can be converted to contain 2-formylglycine (fGly) by the action of 2-formylglycine generating enzyme (FGE). Such a sulfatase motif may also be referred to herein as an FGE modification site. The action of FGE is directed in a sequence-specific manner, in that FGE acts on a sulfatase motif located within an immunoglobulin polypeptide. The moiety of interest is provided as a component of a reactive partner for reaction with the aldehyde of the fGly residue of the converted aldehyde tag of the tagged Ig polypeptide. Attachment of the moiety of interest to the fGly residue of the aldehyde-tagged Ig polypeptide can be achieved using a wide range of commercially available reagents. For example, aminooxy, hydrazide, or thiosemicarbazide derivatives of some moieties of interest are suitable reactive partners and are readily available or can be generated using standard chemical methods.

[0398] As described above, the amino acid sequence of an anti-MUC1 antibody may be modified to contain a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) to a 2-formylglycine (fGly) residue by the action of formylglycine generating enzyme (FGE), either in vivo (e.g., during translation of an aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting an aldehyde-tagged protein with FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

[0399] sulfatase motif The minimum sulfatase motif of the aldehyde tag is typically 5 or 6 amino acid residues in length, and typically no more than 6 amino acid residues in length. The sulfatase motif provided in the Ig polypeptide is at least 5 or 6 amino acid residues and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, defining a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acid residues.

[0400] In certain embodiments, the polypeptide of interest comprises one or more amino acid residues inserted, deleted or substituted (substituted) relative to the native amino acid sequence, for example, two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more, or eleven or more, or twelve or more, or thirteen or more, or fourteen or more, or fifteen or more, or sixteen or more, or seventeen or more, or eighteen or more, or nineteen or more, or twenty or more amino acid residues, to provide the sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide comprises fewer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues altered (inserted, added, deleted, and / or substituted / substituted) in the amino acid sequence compared to the native amino acid sequence of the polypeptide. Where the native amino acid sequence of a polypeptide (e.g., an anti-MUC1 antibody) contains one or more residues of a desired sulfatase motif, the total number of residue modifications can be reduced, for example, by site-specific modification (insertion, addition, deletion, substitution / replacement) of amino acid residues adjacent to the native amino acid residue(s) to provide the desired sulfatase motif sequence. In certain embodiments, the extent of alteration of the native amino acid sequence of the target anti-MUC1 polypeptide is minimized to minimize the number of amino acid residues inserted, deleted, substituted (substituted), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modifications of the target anti-MUC1 polypeptide can minimize the effects that such modifications may have on the function and / or structure of anti-MUC1.

[0401] It should be noted that aldehyde tags of particular interest are those that contain at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), although it is readily understood that longer aldehyde tags are both contemplated and encompassed by the present disclosure and may find use in the compositions and methods of the present disclosure. Thus, an aldehyde tag can contain a minimal sulfatase motif of 5 or 6 residues, or can be longer and contain a minimal sulfatase motif where additional amino acid residues can flank the motif on the N- and / or C-terminal sides. For example, aldehyde tags of 5 or 6 amino acid residues, as well as longer amino acid sequences of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues, are contemplated.

[0402] The aldehyde tag can be located at or near the C-terminus of the Ig heavy chain. For example, the aldehyde tag can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a native wild-type Ig heavy chain. The aldehyde tag can be located in the CH1 domain of the Ig heavy chain. The aldehyde tag can be located in the CH2 domain of the Ig heavy chain. The aldehyde tag can be located in the CH3 domain of the Ig heavy chain. The aldehyde tag can be located in an Ig light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region.

[0403] In certain embodiments, the sulfatase motif used has the formula: X 1 Z 1 X 2 Z 2 X 3 Z 3 (I') wherein: Z 1 is cysteine ​​or serine (can also be represented as (C / S)), Z 2 is either a proline or alanine residue (also represented as (P / A)); Z 3is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), usually A, G, L, V or I, X 1 is present or absent, and if present, can be any amino acid, but typically is an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), typically L, M, V, S, or T, more typically L, M, S, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 can independently be any amino acid, but typically can be an aliphatic amino acid, a polar amino acid, an uncharged amino acid, or a sulfur-containing amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C. For example, S, T, A, V, or G. In one example, the aldehyde tag is of the formula L(C / S)TPSR (SEQ ID NO: 5), e.g., LCTPSR (SEQ ID NO: 6) or LSTPSR (SEQ ID NO: 23). Thus, the present disclosure provides antibodies comprising an aldehyde-tagged Ig heavy chain and / or an aldehyde-tagged Ig light chain, wherein the aldehyde-tagged Ig antibody comprises a heavy and / or light chain Ig constant region amino acid sequence containing such a sulfatase motif.

[0404] For example, in some embodiments, the amino acid sequence of the anti-MUC1 heavy and / or light chain is represented by Formula X 1 Z 1 X 2 Z 2 X 3 Z 3 and can be modified to provide a sequence of at least five amino acids of the formula: Z 1 is cysteine ​​or serine, Z 2 is a proline or alanine residue, Z 3 is an aliphatic amino acid or a basic amino acid, X 1 is present or absent, and if present, is any amino acid, except that when the heterologous sulfatase motif is at the N-terminus of the polypeptide, X 1 exists, X 2 and X 3 are each independently any amino acid, The sequence is within or adjacent to a solvent-accessible loop region of an Ig constant region, and the sequence is not at the C-terminus of an Ig heavy chain.

[0405] The sulfatase motif is generally selected so that it can be converted by a selected FGE, e.g., an FGE present in the host cell in which the aldehyde-tagged polypeptide is expressed, or an FGE that contacts the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0406] For example, when the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including human FGE), the sulfatase motif can be represented by the formula: X 1 CX 2 PX 3 Z 3 (I”) It can be, where: X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 can independently be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; Z3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I.

[0407] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: 6), MCTPSR (SEQ ID NO: 97), VCTPSR (SEQ ID NO: 76), LCSPSR (SEQ ID NO: 77), LCAPSR (SEQ ID NO: 78), LCVPSR (SEQ ID NO: 79), LCGPSR (SEQ ID NO: 80), ICTPAR (SEQ ID NO: 81), LCTPSK (SEQ ID NO: 82), MCTPSK (SEQ ID NO: 83), VCTPSK (SEQ ID NO: 84), LCSPSK (SEQ ID NO: 85), LCAPSK (SEQ ID NO: 86), LCVPSK (SEQ ID NO: 87), LCGPSK (SEQ ID NO: 88), LCTPSA (SEQ ID NO: 89), ICTPAA (SEQ ID NO: 90), MCTPSA (SEQ ID NO: 91), VCTPSA (SEQ ID NO: 92), LCSPSA (SEQ ID NO: 93), LCAPSA (SEQ ID NO: 94), LCVPSA ​​(SEQ ID NO: 95), and LCGPSA (SEQ ID NO: 96).

[0408] fGly-containing sequence Generally, the FGE used to promote the conversion of a cysteine ​​or serine in the sulfatase motif of the aldehyde tag of a target polypeptide to fGly is selected according to the sulfatase motif present in the aldehyde tag. The FGE can be native to the host cell in which the aldehyde-tagged polypeptide is expressed, or the host cell can be genetically modified to express a suitable FGE. In some embodiments, it may be desirable to use a sulfatase motif compatible with human FGE and express the aldehyde-tagged protein in human cells expressing FGE or in host cells, usually mammalian cells, genetically modified to express human FGE. In general, FGEs suitable for use in generating fGly-modified antibodies can be obtained from naturally occurring sources or synthetically produced. For example, suitable FGEs can be derived from organisms that naturally produce FGE or that have been genetically modified to express a recombinant gene encoding FGE. Nucleic acids encoding several FGEs are known and readily available in the art.

[0409] After the action of FGE on the sulfatase motif, Z 1 is oxidized to generate a 2-formylglycine (fGly) residue. Furthermore, after both the FGE-mediated conversion and reaction with a reactive partner containing the moiety of interest, Z in the above formula 1 The fGly position in is covalently attached to a moiety of interest (e.g., a detectable label, a water-soluble polymer, a polypeptide, a drug, an active agent, etc.). Accordingly, the present disclosure provides anti-MUC1 antibodies having amino acid sequences modified to include an fGly moiety.

[0410] Upon action of FGE on the anti-MUC1 heavy and / or light chains, the serine or cysteine ​​in the sulfatase motif is modified to fGly. Thus, the fGly-containing sulfatase motif has the formula: X 1 (fGly)X 2 Z 2 X 3 Z 3 (I"') where: fGly is a formylglycine residue, Z 2 is either a proline or alanine residue (also represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), e.g., A, G, L, V or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 can independently be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0411] As described above, to produce a conjugate, a polypeptide containing an fGly residue can be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety of a linker attached to the drug or active agent (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety described above) to produce an fGly'-containing sulfatase motif. As used herein, the term fGly' refers to an amino acid residue of a sulfatase motif attached to a drug or active agent via a linker described herein. Accordingly, the present disclosure provides anti-MUC1 antibody conjugates (also referred to herein as "anti-MUC1 conjugates").

[0412] In certain embodiments, the anti-MUC1 conjugate has the following formula: X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) a fGly'-containing sulfatase motif of where: fGly' is an amino acid residue attached to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (also represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), e.g., A, G, L, V or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 can independently be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0413] In certain embodiments, the sequence of formula (III) is located at the C-terminus of the heavy chain constant region of the anti-MUC1 antibody. In some examples, the heavy chain constant region has the sequence of formula (III): X 1 (fGly')X 2 Z 2 X 3 Z3 (III) contains an array of where: fGly' is an amino acid residue attached to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (also represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine ...

Claims

1. Formula (I): 【Chemistry 1】 A conjugate of During the ceremony, Z is CR ４ or N, R １ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R ２ and R ３ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R ２ and R ３ may be cyclically linked to form a 5- or 6-membered heterocyclyl; Each R ４ is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W １ is a drug, W ２ is an anti-MUC1 antibody; A conjugate of formula (I).

2. L, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ - Including, During the ceremony, a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is 1 to 6; T １ , T ２ , T ３ , T ４ , T ５ and T ６ each independently represents a covalent bond, (C １ ~C １２ ) alkyl, substituted (C １ ~C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｍ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetals, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12; V １ , V ２ , V ３ , V ４ , V ５ and V ６ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ - and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; The conjugate of claim 1.

3. T １ However, (C １ ~C １２ ) alkyl and substituted (C １ ~C １２ ) alkyl; T ２ , T ３ , T ４ , T ５ and T ６ each independently represents a covalent bond, (C １ ~C １２ ) alkyl, substituted (C １ ~C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｍ -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines and esters; V １ , V ２ , V ３ , V ４ , V ５ and V ６ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-; During the ceremony, (PEG) ｎ but 【Chemistry 2】 where n is an integer from 1 to 30; EDA has the following structure: 【Chemistry 3】 where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP), 【Chemistry 4】 and Each R １２ are independently selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, aryl, and substituted aryl, where any two adjacent R １２ The groups may be cyclically linked to form a piperazinyl ring. The conjugate of claim 2.

4. The conjugate of any one of claims 2 to 3, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

5. 5. The conjugate of claim 4, wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

6. During the ceremony, T １ But (C １ ~C １２ ) alkyl, V １ is -CO-, T ２ is an amino acid analogue, V ２ is -NH-, T ３ (PEG) ｎ And V ３ is -CO-, T ４ is AA, and V ４ does not exist, T ５ is PABC, and V ５ does not exist, f is 0; The conjugate according to any one of claims 2 to 5.

7. The conjugate of any one of claims 1 to 6, wherein the drug is monomethylauristatin E (MMAE).

8. The conjugate has the structure: 【Chemistry 5】 The conjugate according to any one of claims 1 to 7, having the formula:

9. Formula (II): 【Chemistry 6】 A conjugate of During the ceremony, Z １ , Z ２ , Z ３ and Z ４ are each independently CR ２４ , N and C-L Ｂ -W １２ and at least one Z １ , Z ２ , Z ３ and Z ４ is C-L Ｂ -W １２ and R ２１ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R ２２ and R ２３ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R ２２ and R ２３ may be cyclically linked to form a 5- or 6-membered heterocyclyl; Each R ２４ is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L Ａ is the first linker, L Ｂ is a second linker, W １１ is the first drug, W １２ is a second drug, W １３ is an anti-MUC1 antibody; A conjugate of formula (II).

10. Z １ is CR ２４ The conjugate of claim 9,

11. Z １ The conjugate of claim 9 , wherein is N.

12. Z ３ is C-L Ｂ -W １２ The conjugate of claim 9,

13. L Ａ but, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ - Including, a, b, c, d, e and f are each independently 0 or 1; T １ , T ２ , T ３ , T ４ , T ５ and T ６ each independently represents a covalent bond, (C １ ~C １２ ) alkyl, substituted (C １ ~C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V １ , V ２ , V ３ , V ４ , V ５ and V ６ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ - and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; A conjugate according to any one of claims 9 to 12.

14. 14. The conjugate of claim 13, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

15. The conjugate of claim 14, wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

16. During the ceremony, T １ But (C １ ~C １２ ) alkyl, V １ is -CONH-, T ２ is substituted (C １ ~C １２ ) alkyl, V ２ is -CO-, T ３ is AA, and V ３ does not exist, T ４ is PABC, and V ４ does not exist, e and f are each 0; A conjugate according to any one of claims 9 to 15.

17. L Ｂ but, -(T ７ -V ７ ) ｇ -(T ８ -V ８ ) ｈ -(T ９ -V ９ ) ｉ -(T １０ -V １０ ) ｊ -(T １１ -V １１ ) ｋ -(T １２ -V １２ ) ｌ -(T １３ -V １３ ) ｍ -、 During the ceremony, g, h, i, j, k, 1, and m are each independently 0 or 1; T ７ , T ８ , T ９ , T １０ , T １１ , T １２ and T １３ each independently represents a covalent bond, (C １ ~C １２ ) alkyl, substituted (C １ ~C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides, and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V ７ , V ８ , V ９ , V １０ , V １１ , V １２ and V １３ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ - and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; A conjugate according to any one of claims 9 to 16.

18. 18. The conjugate of claim 17, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

19. The conjugate of any one of claims 17 to 18, wherein the glycoside is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc and O-GalNAc.

20. T ７ does not exist, and V ７ is -NHCO-, T ８ However, (C １ ~C １２ ) alkyl, V ８ is -CONH-, T ９ is substituted (C １ ~C １２ ) alkyl, V ９ is -CO-, T １０ is AA, and V １０ does not exist, T １１ is PABC, and V １１ does not exist, l and m are each 0; A conjugate according to any one of claims 17 to 19.

21. The conjugate has the structure: 【Chemistry 7】 10. The conjugate of claim 9, having the formula:

22. The conjugate according to any one of claims 1 to 21, wherein the anti-MUC1 antibody is an IgG1 antibody.

23. The conjugate of claim 22, wherein the anti-MUC1 antibody is an IgG1 kappa antibody.

24. The anti-MUC1 antibody has the formula (III): X １ (fGly’)X ２ Z ２ X ３ Z ３ (III) Contains an array of During the ceremony, X １ is present or absent, and if present, may be any amino acid, except that when said sequence is at the N-terminus of the conjugate, X １ exists, fGly' is the amino acid residue attached to the drug via the linker; X ２ and X ３ are each independently any amino acid; Z ２ is either a proline or an alanine residue, Z ３ is a basic amino acid or an aliphatic amino acid; A conjugate according to any one of claims 1 to 23.

25. 25. The conjugate of claim 24, wherein the sequence is L(fGly')TPSR (SEQ ID NO:24).

26. Z ３ is selected from R, K, H, A, G, L, V, I, and P; X １ is selected from L, M, S and V; X ２ and X ３ are each independently selected from S, T, A, V, G and C; 25. The conjugate of claim 24.

27. The conjugate according to any one of claims 1 to 26, wherein said sequence is located at the C-terminus of the heavy chain constant region of said anti-MUC2 antibody.

28. The heavy chain constant region has formula (III): X １ (fGly’)X ２ Z ２ X ３ Z ３ (III) Contains an array of During the ceremony, X １ is present or absent, and if present, may be any amino acid, except that when said sequence is at the N-terminus of the conjugate, X １ exists, fGly' is the amino acid residue attached to the drug via the linker; X ２ and X ３ are each independently any amino acid; Z ２ is either a proline or an alanine residue, Z ３ is a basic amino acid or an aliphatic amino acid, wherein the sequence is the C-terminus of the amino acid sequence SLSLSPG (SEQ ID NO: 187); 28. The conjugate of claim 27.

29. 29. The conjugate of claim 28, wherein the heavy chain constant region comprises the sequence SPGSL(fGly')TPSRGS (SEQ ID NO: 188).

30. Z ３ is selected from R, K, H, A, G, L, V, I, and P; X １ is selected from L, M, S and V; X ２ and X ３ are each independently selected from S, T, A, V, G and C; 29. The conjugate of claim 28.

31. 31. The conjugate of any one of claims 27 to 30, wherein the heavy chain constant region of the anti-MUC1 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and comprises an fGly' residue at amino acid position 332 instead of C.

32. 27. The conjugate of any one of claims 1 to 26, wherein the heavy chain constant region of the anti-MUC1 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 58, 62, 66, and 70, and comprises a fGly' residue at amino acid position 59 instead of C.

33. 27. The conjugate of any one of claims 1 to 26, wherein the heavy chain constant region of the anti-MUC1 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 59, 63, 67, and 71 and comprises a fGly' residue at amino acid position 62 instead of C.

34. 27. The conjugate of any one of claims 1 to 26, wherein the heavy chain constant region of the anti-MUC1 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 60, 64, 68, and 72 and comprises a fGly' residue at amino acid position 92 instead of C.

35. 27. The conjugate of any one of claims 1 to 26, wherein the heavy chain constant region of the anti-MUC1 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61, 65, 69, and 73 and comprises an fGly' residue at amino acid position 117 instead of C.

36. The conjugate of any one of claims 1 to 26, wherein the fGly' residue is located in the light chain constant region of the anti-MUC1 antibody.

37. The light chain constant region has formula (III): X １ (fGly’)X ２ Z ２ X ３ Z ３ (III) Contains an array of During the ceremony, X １ is present or absent, and if present, may be any amino acid, except that when said sequence is at the N-terminus of the conjugate, X １ exists, fGly' is the amino acid residue attached to the drug via the linker; X ２ and X ３ are each independently any amino acid; Z ２ is either a proline or an alanine residue, Z ３ is a basic amino acid or an aliphatic amino acid, The sequence is C-terminal to the amino acid sequence KVDNAL (SEQ ID NO: 101) and / or N-terminal to the sequence QSGNSQ (SEQ ID NO: 102); 37. The conjugate of claim 36.

38. 38. The conjugate of claim 37, wherein the light chain constant region comprises the sequence KVDNAL(fGly')TPSRQSGNSQ (SEQ ID NO: 103).

39. Z ３ is selected from R, K, H, A, G, L, V, I, and P; X １ is selected from L, M, S and V; X ２ and X ３ are each independently selected from S, T, A, V, G and C; 37. The conjugate of claim 36.

40. The conjugate of any one of claims 1 to 26, wherein said fGly' residue is located in the heavy chain CH1 region of said anti-MUC1 antibody.

41. The light chain constant region has formula (III): X １ (fGly’)X ２ Z ２ X ３ Z ３ (III) Contains an array of During the ceremony, X １ is present or absent, and if present, may be any amino acid, except that when said sequence is at the N-terminus of the conjugate, X １ exists, fGly' is the amino acid residue attached to the drug via the linker; X ２ and X ３ are each independently any amino acid; Z ２ is either a proline or an alanine residue, Z ３ is a basic amino acid or an aliphatic amino acid, The sequence is C-terminal to the amino acid sequence SWNSGA (SEQ ID NO: 104) and / or N-terminal to the amino acid sequence GVHTFP (SEQ ID NO: 105); 41. The conjugate of claim 40.

42. 42. The conjugate of claim 41, wherein the heavy chain CH1 region comprises the sequence SWNSGAL(fGly')TPSRGVHTFP (SEQ ID NO: 106).

43. Z ３ is selected from R, K, H, A, G, L, V, I, and P; X １ is selected from L, M, S and V; X ２ and X ３ are each independently selected from S, T, A, V, G and C; 43. The conjugate of claim 42.

44. The conjugate of any one of claims 1 to 26, wherein said fGly' residue is located in the heavy chain CH2 region of said anti-MUC1 antibody.

45. The conjugate of any one of claims 1 to 26, wherein said fGly' residue is located in the heavy chain CH3 region of said anti-MUC1 antibody.

46. The anti-MUC1 antibody, a variable heavy (VH) chain comprising a VH chain heavy chain CDR1-3 (HCDR1-3) having the sequence of: EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRPGKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSSLRSEDTAVYYCARGLRYALDYWGQGTLVTVSS (SEQ ID NO: 1); EIVLTQSPATHLSPGERATLSCRASSSVSSSSYLYWYQQKPGQAPRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYAWSPPTFGQGTKLEIK (SEQ ID NO:2); EIVLTQSPATHLSPGERATLSCRASSSVGSS NLYWYQQKPGQAPRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYRWSPPTFGQGTKLEIK (SEQ ID NO: 3); or a variable light (VL) chain comprising a VL chain light chain CDR1-3 (LCDR1-3) having the sequence of: EIVLTQSPATHLSPGERATLSCRASSSSVSSSYLYWYQQKPGQAPRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYSWSPPTFGQGTKLEIK (SEQ ID NO: 4); 46. ​​The conjugate of any one of claims 1 to 45, which competes for binding to MUC1 with an anti-MUC1 antibody comprising:

47. The anti-MUC1 antibody, a variable heavy (VH) chain comprising a VH chain heavy chain CDR1-3 (HCDR1-3) having the sequence of: EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRPGKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSSLRSEDTAVYYCARGLRYALDYWGQGTLVTVSS (SEQ ID NO: 1); EIVLTQSPATHLSPGERATLSCRASSSVSSSSYLYWYQQKPGQAPRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYAWSPPTFGQGTKLEIK (SEQ ID NO:2); EIVLTQSPATHLSPGERATLSCRASSSVGSS NLYWYQQKPGQAPRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYRWSPPTFGQGTKLEIK (SEQ ID NO: 3); or a variable light (VL) chain comprising a VL chain light chain CDR1-3 (LCDR1-3) having the sequence of: EIVLTQSPATHLSPGERATLSCRASSSSVSSSYLYWYQQKPGQAPRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYSWSPPTFGQGTKLEIK (SEQ ID NO: 4); The conjugate of any one of claims 1 to 45, comprising:

48. 48. The conjugate of claim 46 or 47, wherein the VH polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:

1.

49. The conjugate according to any one of claims 46 to 48, wherein the VL polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 2, 3 or 4.

50. According to Kabat's definition, the HCDR1 comprises the amino acid sequence DHTMH (SEQ ID NO: 17); the HCDR2 comprises the amino acid sequence YFYPRDDSTNYNEKFKG (SEQ ID NO: 18); the HCDR3 comprises the amino acid sequence GLRYALDY (SEQ ID NO:9); the LCDR1 comprises the amino acid sequence RASSSVSSSYLY (SEQ ID NO: 10); the LCDR2 comprises the amino acid sequence GTSNLAS (SEQ ID NO: 11); the LCDR3 comprises the amino acid sequence HQYAWSPPT (SEQ ID NO: 12); 50. The conjugate according to any one of claims 46 to 49.

51. According to Kabat's definition, the HCDR1 comprises the amino acid sequence DHTMH (SEQ ID NO: 17); the HCDR2 comprises the amino acid sequence YFYPRDDSTNYNEKFKG (SEQ ID NO: 18); the HCDR3 comprises the amino acid sequence GLRYALDY (SEQ ID NO:9); the LCDR1 comprises the amino acid sequence RASSSVGSSNLY (SEQ ID NO: 13); the LCDR2 comprises the amino acid sequence RSTKLAS (SEQ ID NO: 14); the LCDR3 comprises the amino acid sequence HQYRWSPPT (SEQ ID NO: 15); 50. The conjugate according to any one of claims 46 to 49.

52. According to Kabat's definition, the HCDR1 comprises the amino acid sequence DHTMH (SEQ ID NO: 17); the HCDR2 comprises the amino acid sequence YFYPRDDSTNYNEKFKG (SEQ ID NO: 18); the HCDR3 comprises the amino acid sequence GLRYALDY (SEQ ID NO:9); the LCDR1 comprises the amino acid sequence RASSSVSSSYLY (SEQ ID NO: 10); the LCDR2 comprises the amino acid sequence GTSNLAS (SEQ ID NO: 11); the LCDR3 comprises the amino acid sequence HQYSWSPPT (SEQ ID NO: 16); 50. The conjugate according to any one of claims 46 to 49.

53. A conjugate according to any one of claims 1 to 52, A pharma- ceutically acceptable excipient; 13. A pharmaceutical composition comprising:

54. A method comprising administering to a subject an effective amount of a conjugate according to any one of claims 1 to 52.

55. 1. A method of treating cancer in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 53, wherein said administering is effective to treat cancer in the subject. A method of treating cancer in a subject.

56. 56. The method of claim 55, wherein the cancer is breast cancer, ovarian cancer, lung cancer, or gastric cancer.

57. 57. The method of claim 56, wherein the cancer is characterized by cancer cells that express glycosylated MUC1.

58. 57. The method of claim 56, wherein the conjugate binds to the glycosylated MUC1.

59. The method of any one of claims 55 to 58, wherein the breast cancer is triple negative for estrogen, progesterone and HER2.

60. 60. The method of claim 59, wherein the triple-negative breast cancer is metastatic triple-negative breast cancer.

61. 61. The method of claim 59 or 60, wherein the triple-negative breast cancer is recurrent or refractory triple-negative breast cancer.

62. 1. A method for delivering a drug to a target site in a subject, comprising:

54. The method of claim 53, comprising administering to the subject the pharmaceutical composition of claim 53, wherein said administering is effective to release a therapeutically effective amount of said drug from said conjugate at said target site in said subject. A method for delivering a drug to a target site in a subject.

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