Mucin-1 specific antibody conjugates and methods of use thereof

Abstract

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

Description

[0001] Cross - reference to related patent applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 227,666, filed Jul. 30, 2021, U.S. Provisional Application No. 63 / 322,914, filed Mar. 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.

[0003] Incorporation by reference of electronically submitted materials

[0004] The Sequence Listing is provided herein as a Sequence Listing XML, “RDWD - 045WO_SEQ_LIST” created on Oct. 13, 2022 and having a size of 164 KB. The content of the Sequence Listing XML is incorporated herein by reference in its entirety. Background of the Invention

[0005] The field of protein - small molecule therapeutic conjugates has made great progress, providing many clinically beneficial drugs and promising to provide more in the coming years. Protein - conjugate therapeutic agents can offer multiple advantages, e.g., fewer side effects due to specificity, functional diversity, and relatively low off - target activity. Chemical modification of proteins can extend these advantages by making them more effective, more stable, or more multimodal.

[0006] Many standard chemical transformations are commonly used to generate and manipulate post - translational modifications of proteins. There are many ways to selectively modify the side chains of certain amino acids. For example, carboxylic acid side chains (aspartic acid and glutamic acid) can be targeted by initial activation with a water - soluble carbodiimide reagent and subsequent reaction with an amine. Similarly, lysine can be targeted by using an activated ester or isothiocyanate, and cysteine thiols can be targeted by maleimide and α - halo carbonyls.

[0007] A major obstacle in inventing chemically altered protein therapeutic agents or reagents is to produce the protein in a biologically active, homogeneous form. The conjugation of a drug or detectable label to a polypeptide can be difficult to control, resulting in a heterogeneous mixture of conjugates with different numbers of attached drug molecules and chemical conjugation positions. In some cases, it is 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.

[0008] Mucin-1 (also known as mucin 1 or MUC1) is a member of the mucin family. Mucins are O-glycosylated proteins that play an important role in forming a protective mucus barrier on epithelial surfaces. MUC1 is expressed on the apical surface of epithelial cells, which line the mucosal surfaces of many different tissues, including the lung, breast, stomach, and pancreas. This protein is proteolytically cleaved into α and β subunits 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 of this protein, abnormal intracellular localization, and alterations in glycosylation are associated with cancer.

[0009] There is a need in the art for safe and effective agents that target MUC1 for the treatment of MUC1-related disorders, such as cancer. SUMMARY OF THE INVENTION

[0010] The present disclosure provides antibody conjugates specific for MUC1 (e.g., antibody-drug conjugates (ADCs)). The present disclosure also includes methods for producing such conjugates, as well as methods of using them. Each embodiment is described in more detail below. Compositions comprising the ADCs of the present disclosure are also provided, including, in some cases, pharmaceutical compositions. In certain aspects, methods of using the ADCs are provided, including administering a therapeutically effective amount of the ADCs of the present disclosure to an individual having a cell proliferative disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is shown that the anti-MUC1 antibodies MUC1gB06, MUC1 G12, and MUC1 H02 are greater than 99%, greater than 99%, and greater than 98% monomer, respectively, as determined by size exclusion chromatography (SEC).

[0012] Figures 2A - 2C It is shown that the anti-MUC1 antibodies MUC1 gB06, MUC1 G12, and MUC1H02 bind to recombinant 20mer MUC1 glycosylated biotin, but not to recombinant 60mer MUC1 non-glycosylated biotin or a bait peptide, as evaluated by ELISA.

[0013] Figures 3A - 3B It is shown the binding levels of the anti-MUC1 antibodies MUC1 gB06, MUC1 G12, and MUC1H02 to uncoated streptavidin or Maxisorp plates.

[0014] Figure 4 It is shown overlay histograms demonstrating binding of the indicated antibodies to the named cell lines, tested in triplicate.

[0015] Figure 5 It is shown staggered histograms demonstrating binding of the indicated antibodies to the named cell lines.

[0016] Figure 6 Shows the melting temperatures of the CH2 and Fab regions of B06, G12, and H02 anti-MUC1 antibodies determined by differential scanning fluorimetry.

[0017] Figure 7 : Production of aldehyde-labeled antibodies and generation of ADCs using HIPS-mediated conjugation. (A) The formylglycine recognition sequence (CXPXR) was genetically encoded into the antibody. (B) Cotranslational formylglycine-generating enzyme converts the cysteine within the recognition sequence into a formylglycine residue containing an aldehyde functional group that can specifically conjugate with (C) a hydrazino-iso-Pictet-Spengler (HIPS) conjugation element.

[0018] Figure 8 : As determined by HIC, the drug-to-antibody ratio (DAR) of the CT-labeled B06 antibody conjugated with RED-601 was 1.85.

[0019] Figure 9 : As determined by SEC, the CT-labeled B06 antibody conjugated with RED-601 was 99.3% monomeric.

[0020] Figure 10 : As determined by HIC, the DAR of the CT-labeled G12 antibody conjugated with RED-601 was 1.89.

[0021] Figure 11 : As determined by SEC, the CT-labeled G12 antibody conjugated with RED-601 was 99.9% monomeric.

[0022] Figure 12 : As determined by HIC, the DAR of the CT-labeled H02 antibody conjugated with RED-601 was 1.90.

[0023] Figure 13 : As determined by SEC, the CT-labeled H02 antibody conjugated with RED-601 was 99.1% monomeric.

[0024] Figure 14 : In vitro potency of monomethyl auristatin E (MMAE)-conjugated anti-MUC1 ADCs made from B06, G12, or H02 variant antibodies against T47D cells. Free MMAE was included as a benchmark for payload potency.

[0025] Figure 15: In vivo efficacy of MMAE-conjugated anti-MUC1 ADCs-B06 RED-601 and H02 RED-601 carrying the MMAE payload. n = 10 mice / group; dosing is indicated by arrows.

[0026] Figure 16 : Representative data showing binding of B06 ADC to primary human ovarian adenocarcinomas. Four ovarian adenocarcinoma specimens were reacted with B06 ADC (top row) or isotype control ADC (bottom row). Brown indicates ADC binding.

[0027] Figure 17 : Representative data showing binding of B06 ADC to primary human lung tumors. Four lung cancer samples were reacted with B06 ADC (top row) or isotype control ADC (bottom row). The left two columns are adenocarcinomas; the right two columns are squamous cell carcinomas. Brown indicates ADC binding.

[0028] Figure 18 : Representative data showing binding of B06 ADC to primary human breast tumors. Four breast ductal carcinoma specimens were reacted with B06 ADC (top row) or isotype control ADC (bottom row). Brown indicates ADC binding.

[0029] Figure 19 : B06 ADC binds strongly to patient-derived xenograft (PDX) tumor models. Four Charles River Laboratories PDX samples were reacted with B06 ADC (top row) or isotype control ADC (bottom row). Brown indicates ADC binding. Tumor origin from left to right: stomach, breast, lung, stomach.

[0030] Figure 20 : Structure of RED-601, which is a linker-payload conjugated to an anti-MUC1 antibody (see Compound 8 in Example 2).

[0031] Figure 21A A site map showing possible modification sites for generating aldehyde-labeled Ig polypeptides is shown. The upper sequence is the amino acid sequence of the conserved region of the IgG1 light chain polypeptide (SEQ ID NO: 74) and shows possible modification sites in the Ig light chain; the lower sequence is the amino acid sequence of the conserved region of the Ig heavy chain polypeptide (SEQ ID NO: 75) (GenBank accession number AAG00909) and shows possible modification sites in the Ig heavy chain. Heavy and light chain numbering is based on the full-length heavy and light chains.

[0032] Figures 21B - 21CAlignment of the constant regions of human immunoglobulin heavy chains 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) is shown, showing modification sites that can provide aldehyde tags in immunoglobulin heavy chains. Heavy and light chain numbering is based on the complete heavy and light chains.

[0033] Figure 21D Alignment of the constant regions of immunoglobulin light chains is shown, showing modification sites that can provide aldehyde tags in immunoglobulin light chains. Seq1 = human κ light chain constant region; GenBank CAA75031.1; SEQ ID NO: 52. Seq2 = human κ light chain constant region; GenBank BAC0168.1; SEQ ID NO: 53. Seq3 = human λ 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.

[0034] Figure 22 : Binding of anti-MUC1 affinity matured variant B06 and control antibodies, PankoMab and 1B2, to 20mer MUC1 glycosylated biotinylated peptide as evaluated by ELISA.

[0035] Figure 23 : Binding of anti-MUC1 affinity matured variants G12 and H02 to 20mer MUC1 glycosylated biotinylated peptide as evaluated by ELISA.

[0036] Figure 24 : Binding of anti-MUC1 affinity matured variant B06 and control antibodies, PankoMab and 1B2, to 60mer MUC1 non-glycosylated biotinylated peptide as evaluated by ELISA.

[0037] Figure 25 : Binding of anti-MUC1 affinity matured variants G12 and H02 to 60mer MUC1 non-glycosylated biotinylated peptide as evaluated by ELISA.

[0038] Figure 26 : Binding of anti-MUC1 affinity matured variants and parental antibodies to antigen-positive T47D cells or antigen-negative HEK cells as evaluated by flow cytometry.

[0039] Figure 27 : In vitro potency of maytansine or monomethyl auristatin E (MMAE)-conjugated anti-MUC1 ADCs made with B06 or H02 variant antibodies against UACC-812 cells. Free maytansine was included as a benchmark for payload potency.

[0040] Figure 28 : As determined by SEC, the single-labeled B06 antibody conjugated with branched MMAE linker-payload at 91N was 96.4% monomer.

[0041] Figure 29 : As determined by HIC, the single-labeled B06 antibody conjugated with compound 8 at 91N yielded a DAR of 1.78.

[0042] Figure 30 : As determined by SEC, the single-labeled B06 antibody conjugated with compound 8 at 91N was 96.2% monomer.

[0043] Figure 31 : As determined by PLRP, the single-labeled B06 antibody conjugated with compound 21 at 91N yielded a DAR of 3.74.

[0044] Figure 32 : As determined by SEC, the single-labeled B06 antibody conjugated with compound 21 at 91N was 95.9% monomer.

[0045] Figure 33 : As determined by PLRP, the double-labeled B06 antibody conjugated with compound 21 yielded a DAR of 7.47.

[0046] Figure 34 : As determined by SEC, the double-labeled B06 antibody conjugated with compound 21 was 96.7% monomer.

[0047] Definition

[0048] "Alkyl" refers to a monovalent saturated aliphatic alkyl group having from 1 to 10 carbon atoms, such as from 1 to 6 carbon atoms, or from 1 to 5, or from 1 to 4, or from 1 to 3 carbon atoms. For example, the term includes straight-chain and branched hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0049] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms (except C1 carbon atoms) on the alkyl chain are optionally substituted by a heteroatom, such as -O-, -N-, -S-, -S(O) n -(where n is from 0 to 2), -NR- (where R is hydrogen or alkyl), and having from 1 to 5 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, amido, aminoacyloxy, oxyamido, azido, cyano, halogen, hydroxy, oxo, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b , where R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic.

[0050] "Alkylene" refers to a divalent aliphatic hydrocarbon group, preferably having from 1 to 6 and more preferably from 1 to 3 carbon atoms, which is straight-chain or branched and is optionally selected from -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10interrupted by one or more groups such as. For example, the term includes methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-, -C(CH3)2CH2CH2-, -C(CH3)2CH2C(O)-, -C(CH3)2CH2C(O)NH-, -CH(CH3)CH2-), etc.

[0051] "Substituted alkylene" means an alkylene having 1 to 3 hydrogens substituted by substituents, as described for carbon in the definition of "substituted" below.

[0052] The term "alkane" refers to alkyl and alkylene as defined herein.

[0053] The terms "alkylaminoalkyl", "alkylaminoalkenyl" and "alkylaminoalkynyl" refer to the group R’NHR” - where R’ is an alkyl as defined herein, and R” is an alkylene, alkenylene or alkynylene group as defined herein.

[0054] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene and aralkyl are defined herein.

[0055] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, etc. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O- and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl and alkynyl are as defined herein.

[0056] The term "substituted alkoxy" refers to a group substituted by alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O- and alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0057] The term "alkoxyamino" refers to the -NH-alkoxy group, where alkoxy is as defined herein.

[0058] The term "haloalkoxy" refers to an alkyl-O-group in which one or more hydrogen atoms on the alkyl group are replaced by halogen groups, and includes groups such as trifluoromethoxy.

[0059] The term "haloalkyl" refers to a substituted alkyl group as described above, in which one or more hydrogen atoms on the alkyl group are replaced by halogen groups. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0060] The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0061] The term "alkylthioalkoxy" refers to the groups -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0062] "Alkenyl" refers to a straight-chain or branched hydrocarbon group 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. For example, the term includes butadienyl, allyl, and but-3-en-1-yl. The term includes cis and trans isomers or mixtures of these isomers.

[0063] The term "substituted alkenyl" refers to an alkenyl group as defined herein having 1 to 5 substituents, or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, amido, amidoxy, oxyamido, azide, cyano, halogen, hydroxy, oxo, thioxo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0064] "Alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1 and preferably 1 to 2 triple-bond unsaturated sites. Examples of such alkynyl groups include ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0065] The term "substituted alkynyl" refers to an alkynyl group as defined herein having 1 to 5 substituents, or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, amido, amidoxy, oxyamido, azido, cyano, halogen, hydroxy, oxo, thioxo, carboxy, carboxyalkyl, thioaryloxy, thioheteraryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0066] "Alkynyloxy" refers to the group -O-alkynyl, where alkynyl is as defined herein. Alkynyloxy includes, for example, ethynyloxy, propynyloxy, and the like.

[0067] "Acyl" refers to the groups H-C(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)-, heterocyclic-C(O)-, and substituted heterocyclic-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)-.

[0068] "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 20C(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 20 is hydrogen or alkyl, and wherein 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] "Aminocarbonyl" or the term "aminoacyl" refers to the group -C(O)NR 51 R 52 wherein R 51 and R 52 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, and wherein R 51 and R 52 optionally combine with the nitrogen to which they are attached to form a heterocyclic or substituted heterocyclic group, wherein 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.

[0070] "Aminocarbonylamino" refers to the group -NR 51 C(O)NR 52 R 53 wherein R 51 、R 52 and R 53 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or wherein two R groups combine together to form a heterocyclic group.

[0071] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl or heterocyclic group, and alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic group are as defined herein.

[0072] 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 heterocyclic group-C(O)O-, and alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic group are as defined herein.

[0073] "Aminosulfonyl" refers to the group -SO2NR 51 R 52 where R 51 and R 52 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, and where R 51 and R 52 optionally combine with the nitrogen to which they are attached to form a heterocyclic or substituted heterocyclic group, and 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.

[0074] "Sulfonylamino" refers to the group -NR 51 SO2R 52 where R 51 and R 52 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, and where R 51 and R 52 optionally combine with the atom 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, heterocycle and substituted heterocycle are as defined herein.

[0075] "Aryl" or "AR" means a monovalent aromatic carbocyclic group having 6 to 18 carbon atoms, which group has a single ring (such as present in phenyl) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracenyl, and indanyl), the fused rings of which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring. The term includes, for example, phenyl and naphthyl. Unless otherwise restricted by the definition of the aryl substituent, these aryl groups may optionally be substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, mercapto, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, amido, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic group, heterocyclic oxy group, amidooxy, 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.

[0076] "Aryloxy" means the group -O-aryl, where aryl is as defined herein, and includes, for example, phenoxy, naphthyloxy, etc., including optionally substituted aryl groups as defined herein.

[0077] "Amino" means the group -NH2.

[0078] The term "substituted amino" means the group -NRR, where each R is independently selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic group, provided that at least one R is not hydrogen.

[0079] The term "azido" means the group -N3.

[0080] "Carboxyl", "carboxy" or "carboxylate" means -CO2H or its salts.

[0081] "Carboxyl ester" or "carboxy ester" or the term "carboxyalkyl" or "carboxylalkyl" refers 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-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocycle and -C(O)O-substituted heterocycle, wherein 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.

[0082] "(Carboxyl ester)oxy" or "carbonate" refers to the groups -O-C(O)O-alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O-C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O-C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O-substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O-heterocycle and -O-C(O)O-substituted heterocycle, wherein 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.

[0083] "Cyano" or "nitrile" refers to the group -CN.

[0084] "Cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms, having a single or multiple cyclic rings, including fused rings, bridged rings and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. For example, such cycloalkyl includes monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl, etc.

[0085] The term "substituted cycloalkyl" refers to an alkynyl group having 1 to 5 substituents, or 1 to 3 substituents, which substituents are selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thioxo, carboxyl, carboxyalkyl, thioaryloxy, thioheteraryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0086] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group having 3 to 10 carbon atoms, having a single or multiple rings and having at least one double bond and preferably 1 to 2 double bonds.

[0087] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents, or 1 to 3 substituents, which substituents are selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thioxo, carboxyl, carboxyalkyl, thioaryloxy, thioheteraryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0088] "Cycloalkynyl" refers to a non-aromatic cycloalkyl having 5 to 10 carbon atoms, having a single ring or multiple rings and having at least one triple bond.

[0089] "Cycloalkoxy" refers to -O-cycloalkyl.

[0090] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0091] "Halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0092] "Hydroxy" or "hydroxyl" refers to the group -OH.

[0093] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms within the ring, which are selected from oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (e.g., pyridyl, imidazolyl, or furyl) or multiple fused rings (e.g., in groups such as indolizinyl, quinolinyl, benzofuranyl, benzimidazolyl, or benzothiophenyl) in the ring system, where at least one ring in the ring system is aromatic. To satisfy valence requirements, any heteroatom in such heteroaryl rings may or may not be bonded to H or a substituent, such as an alkyl group or other substituents described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. For example, the term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl, and furyl. Unless the definition of the heteroaryl substituent is otherwise limited, such heteroaryl groups may optionally be substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, mercapto, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, amido, acylamino, alkaryl, aryl, aryloxy, azide, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, amidooxy, oxyamido, 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.

[0094] The term "heteroarylalkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are as defined herein. For example, the term includes pyridylmethyl, pyridylethyl, indolylmethyl, etc.

[0095] "Heteroaryloxy" refers to -O-heteroaryl.

[0096] "Heterocycle", "heterocyclic", "heterocycloalkyl", and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused rings, bridged rings, 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, where, in a fused ring system, one or more rings may 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 atoms of the heterocyclyl are optionally oxidized to provide N-oxide, -S(O)-, or -SO2- moieties. To satisfy valence requirements, any heteroatom in the ring of such a heterocycle may or may not be bonded to one or more H or one or more substituents, such as an alkyl group or other substituents described herein.

[0097] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinazoline, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, 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 known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0098] Unless the definition of the substituent of the heterocycle is otherwise limited, such a heterocyclyl may optionally be substituted with 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, amido, amidoxy, oxyamido, azide, cyano, halogen, hydroxy, oxo, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycles.

[0099] "Heterocyloxy" refers to -O-heterocyclyl.

[0100] The term "heterocyclylthio" refers to the group heterocycle-S-.

[0101] The term "heterocyclene" refers to a diradical group formed from a heterocycle as defined herein.

[0102] The term "hydroxyamino" refers to the group -NHOH.

[0103] "Nitro" refers to the group -NO2.

[0104] "Oxo" refers to the atom (=O).

[0105] "Sulfonyl" refers to the groups SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocycle and SO2-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 includes, for example, methyl-SO2-, phenyl-SO2- and 4-methylphenyl-SO2-.

[0106] "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.

[0107] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl or heterocyclic group, where alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic group are as defined herein.

[0108] "Thiol" refers to the group -SH.

[0109] "Thioxo" or the term "thioketo" refers to the atom (=S).

[0110] "Alkylthio" or the term "thioalkoxy" refers to the group -S-alkyl, where alkyl is as defined herein. In certain embodiments, sulfur can be oxidized to -S(O)-. The sulfoxide can exist as one or more stereoisomers.

[0111] The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

[0112] The term "thioaryloxy" refers to the group aryl-S-, where the aryl group is as defined herein, including optionally substituted aryl groups as also defined herein.

[0113] The term "thioheteroaryloxy" refers to the group heteroaryl-S-, where heteroaryl is as defined herein, including optionally substituted aryl groups as also defined herein.

[0114] The term "thioheterocyclooxy" refers to the group heterocyclo-S-, where the heterocyclo group is as defined herein, including optionally substituted heterocyclo groups as also defined herein.

[0115] Except as disclosed herein, when the term "substituted" is used to modify a particular group or radical, it can also mean that one or more hydrogen atoms of the particular group or radical are independently replaced by the same or different substituents as defined below.

[0116] Except for the groups disclosed for individual terms herein, unless otherwise specified, on a saturated carbon atom of a particular group or radical, substituents used to replace one or more hydrogens (any two hydrogens on a single carbon can be replaced by =O, =NR 70 , =N-OR 70 , =N2 or =S substitution) are -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trifluoromethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R70 、-OSO2O - M + 、-OSO2OR 70 、-P(O)(O - )2(M + )2、-P(O)(OR 70 ) - M + 、-P(O)(OR 70 )2. -C(O)R 70 、-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)OM + 、-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, aralkyl, heteroaryl and heteroaralkyl, each R 70 are independently hydrogen or R 60 ; Each R 80 Independently for R70 Alternatively, two Rs 80 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycloalkyl which may optionally contain 1-4 additional identical or different heteroatoms selected from O, N and S, where N may have -H or C1-C3 alkyl substitution; and each M + is a counterion with a net single positive charge. Each M + may independently be, for example, an alkali ion such as K + , Na + , Li + ; an ammonium ion such as + N(R 60 )4; or an alkaline earth ion such as [Ca 2+ 0.5 , [Mg 2 + 0.5 or [Ba 2+ 0.5 ("The subscript 0.5 means that one counterion of such a divalent alkaline earth ion can be the ionized form of the compound of the present invention, while the other can be a typical counterion such as chloride, or two ionized compounds disclosed herein can be used as the counterions of such a divalent alkaline earth metal ion, or the doubly ionized compound of the present invention can be used as the counterion of such a divalent alkaline earth metal ion). As a specific example, -NR 80 R 80 refers to including -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl).

[0117] Unless otherwise specified, in addition to the content disclosed herein, in "substituted" alkenes, alkynes, aryls and heteroaryls, the substituents on the unsaturated carbon atoms are -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -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 and -NR 70 C(NR 70 ) NR 80 R 80 , where R 60 , R 70 , R 80 and M + are as defined above, provided that in the case of a substituted alkene or alkyne, the substituent is not -O - M + , -OR 70 , -SR 70 or -S - M + .

[0118] Except for the groups disclosed for the individual terms herein, unless otherwise specified, the substituent groups for the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl are -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 70C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 wherein R 60 , R 70 , R 80 and M + are as defined above.

[0119] In addition to the disclosure herein, in one embodiment, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0120] It should be understood that among all the substituents defined above, polymers obtained by defining substituents that themselves have other substituents (e.g., a substituted aryl having a substituted aryl as a substituent that is itself a substituted aryl group, which is further substituted by a substituted aryl, etc.) are not included herein. In this case, the maximum number of such substitutions is three. For example, the series of substitutions of substituted aryls specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0121] Unless otherwise specified, the nomenclature of substituents not explicitly defined herein is obtained by naming the terminal portion of the functional group and then naming the adjacent functional group towards the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-O-C(O)-.

[0122] Regarding any group containing one or more substituents disclosed herein, it should be understood that such a group does not include any substitution or substitution pattern that is not physically and / or synthetically feasible in space. In addition, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

[0123] 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 dosage 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 a pharmaceutically acceptable salt of a compound, which is derived from various organic and inorganic counterions well known in the art and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and when the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, etc.

[0124] The term "salt thereof" refers to a compound formed when the proton of an acid is replaced by a cation (such as a metal cation or an organic cation, etc.). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not the case for salts of intermediate compounds not intended for administration to a patient. By way of example, salts of the compounds of the present invention include those salts in which the compound is protonated by an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid serves as the anionic component of the salt.

[0125] "Solvate" refers to a complex formed by the combination of solvent molecules with the molecules or ions of a solute. 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.

[0126] "Stereoisomer" and "Stereoisomers" refer to compounds having the same atomic connectivity but different atomic arrangements in space. Stereoisomers include cis-trans isomers, E- and Z-isomers, enantiomers, and diastereomers.

[0127] "Tautomer" refers to alternative forms of a molecule that differ only in the electronic bonds and / or proton positions of the atoms, such as enol-ketone and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing the -N=C(H)-NH- ring atom arrangement, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. Those of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are possible.

[0128] It should 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.

[0129] The terms “antibodies” 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 tetramers, which in turn are composed of two dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); antibody fragments that retain specific binding to an antigen (e.g., whole-chain or single-chain antibody fragments), including, but not limited to, Fab, Fv, scFv, 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 using, e.g., radioisotopes, enzymes that produce a detectable product, fluorescent proteins, etc. The antibodies can be further conjugated to other moieties, such as components of specific binding pairs, e.g., biotin (a component of the biotin-avidin specific binding pair), etc. Antibodies can also be bound to solid carriers, including, but not limited to, polystyrene plates or beads, etc. The term also includes Fab’, Fv, F(ab’)2, and / or other antibody fragments that retain specific binding to an antigen, and monoclonal antibodies. Antibodies can be monovalent or divalent.

[0130] “Antibody fragments” include portions of a whole antibody, such as the antigen-binding or variable region of a whole antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Pepsin 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, the name reflecting its ability to crystallize readily. Pepsin treatment yields F(ab’)2 fragments, which have two antigen-binding sites and are still able to cross-link antigens.

[0131] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain in a tight, non-covalent association. It is in this configuration that the three CDRs of 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 the Fv that contains only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0132] The "Fab" fragment also contains the constant region of the light chain and the first constant region (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of some residues at the carboxyl terminus of the heavy-chain CH1 domain, which includes one or more cysteines from the antibody hinge region. Fab'-SH is the name in this text for the Fab' in which the cysteine residue of the constant region bears a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with a hinge-region cysteine between them. Other chemical conjugations of antibody fragments are also known.

[0133] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be grouped into one of two distinct types based on the amino acid sequence of its constant region, called kappa (κ) and lambda (λ). Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy-chain constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into multiple subclasses (isotypes), such as, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0134] The "single-chain Fv" or "sFv" antibody fragment contains the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain. In some aspects, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which enables the sFv to form the structure required for antigen binding.

[0135] The term "diabody" refers to small antibody fragments that have two antigen-binding sites and contain heavy-chain variable domains (V H -V L ) linked to light-chain variable domains (V L ) in the same polypeptide chain (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 domain on the other chain and create two antigen-binding sites.

[0136] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two reagents and is expressed as the dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater or more compared to 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 higher. As used herein, the term "avidity" refers to the resistance of a complex of two or more reagents to dissociation upon dilution. In the context of antibodies and / or antigen-binding fragments, the terms "immunoreactive" and "preferentially bind" are used interchangeably herein.

[0137] The term "binding" refers to a direct association between two molecules, such as due to covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bond interactions, including interactions such as salt bridges and water bridges. A subject anti-MUC1 antibody specifically binds to an epitope within a MUC1 polypeptide, such as a human MUC1 polypeptide, for example, glycosylated MUC1 or a fragment thereof. Nonspecific binding refers to binding with an affinity of less than about 10 -7 M, such as binding with an affinity of 10 -6 M, 10 -5 M, 10 -4 M, etc.

[0138] The term "specifically binds" in the context of an antibody and an antigen means that the antibody binds to or associates with the antigen with an affinity, for example, of greater than or equal to about 10 5 M -1 or a K a (i.e., the equilibrium association constant for a specific binding interaction with units of 1 / M) for the antigen.

[0139] "High affinity" binding means that the K a is at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 M, at least 1010 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or greater association. Alternatively, affinity can be defined as the equilibrium dissociation constant (K D )(e.g., 10 -5 M to 10 -13 M or less). In some embodiments, specific binding refers to the K D of an antibody binding to an antigen being less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, or less than or equal to about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or less. The binding affinity of an antibody for an antigen can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., BIAcore 2000 instrument, using the general procedures outlined by the manufacturer); by radioimmunoassay; etc.

[0140] As used herein, the term “CDR” or “complementary determining region” refers to the non - contiguous antigen - binding sites present within the variable regions of heavy - and light - chain polypeptides. CDRs have been described in Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901 - 917 (1987); and MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996), where the definition includes overlaps or subsets of amino acid residues when compared to each other. However, application of any of these definitions to refer to the CDRs of an antibody or a grafted antibody or its variants is intended to be within the scope of the terms defined and used herein. For comparison, the amino acid residues comprising the CDRs as defined by the references cited above are listed in Table 1 below.

[0141] Table 1: CDR Definitions

[0142] <![CDATA[Kabat 1 > <![CDATA[Chothia 2 > <![CDATA[MacCallum 3 > <![CDATA[V H CDR1]]> 31-35 26-32 30-35 <![CDATA[V H CDR2]]> 50-65 53-55 47-58 <![CDATA[V H CDR3]]> 95-102 96-101 93-101 <![CDATA[V L CDR1]]> 24-34 26-32 30-36 <![CDATA[V L CDR2]]> 50-56 50-52 46-55 <![CDATA[V L CDR3]]> 89-97 91-96 89-96

[0143] 1 The residue numbering follows the nomenclature of Kabat et al. as described above.

[0144] 2 The residue numbering follows the nomenclature of Chothia et al. as described above.

[0145] 3 The residue numbering follows the nomenclature of MacCallum et al. as described above.

[0146] Throughout the present disclosure, the residue numbering in immunoglobulin heavy and light chains is the same as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is hereby expressly incorporated by reference.

[0147] As used herein, when referring to the variable region of an antibody, the term "framework" refers to all amino acid residues outside the CDR regions within the variable region of the antibody. The variable region framework is typically a discontinuous amino acid sequence having a length between about 100 and 120 amino acids, but only those amino acids outside the CDRs are referred to. As used herein, the term "framework region" refers to each domain of the framework separated by the CDRs.

[0148] A "parental Ig polypeptide" is a polypeptide comprising an amino acid sequence that lacks the aldehyde-tagged constant region described herein. The parental polypeptide may comprise a native sequence constant region or may comprise a constant region having pre-existing amino acid sequence modifications (e.g., additions, deletions, and / or substitutions).

[0149] In the context of Ig polypeptides, the term "constant region" is well known in the art and refers to the C-terminal region of an Ig heavy or light chain. The Ig heavy chain constant region includes the CH1, CH2, and CH3 domains (and the CH4 domain, where the heavy chain is a μ or ε heavy chain). In a native Ig heavy chain, the CH1, CH2, CH3 (and CH4 if present) domains immediately follow (C-terminal to) the heavy chain variable (VH) region, and each domain has a length of approximately 100 amino acids to approximately 130 amino acids. In a native Ig light chain, the constant region immediately follows (C-terminal to) the light chain variable region (VL) and has a length of about 100 amino acids to 120 amino acids.

[0150] "Epitope" refers to the site on an antigen to which an antibody binds (e.g., a site on MUC1). Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by protein folding (e.g., tertiary folding). Epitopes formed by contiguous amino acids generally remain upon exposure to denaturing solvents, whereas epitopes formed by folding are generally lost upon treatment with denaturing solvents. Epitopes generally include at least 3, and more typically at least 5 or 8 - 10, amino acids in a linear or conformational arrangement. Methods for determining the conformational structure of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, edited by Glenn E. Morris (1996). Some commercial laboratories offer epitope mapping services. Epitopes bound by antibodies immunoreactive with membrane-associated antigens can be present on the cell surface (e.g., in the extracellular region of a transmembrane protein), and such epitopes are thus considered to be cell surface accessible, solvent accessible, and / or cell surface exposed epitopes.

[0151] When referring to the amino acid sequence of a polypeptide, peptide, or protein, the use of "genetically-encodable" means that the amino acid sequence consists of amino acid residues that can be produced by transcription and translation of a nucleic acid encoding the amino acid sequence, where the transcription and / or translation can occur in a cell or in an in vitro cell-free transcription / translation system.

[0152] The term "control sequences" refers to DNA sequences that promote the expression of an operably linked coding sequence in a particular expression system (e.g., mammalian cells, bacterial cells, cell-free synthesis, etc.). For example, control sequences suitable for prokaryotic systems include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cell systems can utilize a promoter, a polyadenylation signal, and an enhancer.

[0153] A nucleic acid is "operably linked" when it is in 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 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 positioned to promote the initiation of translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader sequence, contiguous and in frame. The linkage is accomplished by ligation or by amplification reactions. Synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice to ligate the sequences.

[0154] As used herein, the term "expression cassette" refers to a nucleic acid fragment, typically DNA, that can be inserted into a nucleic acid (e.g., by using restriction sites compatible with those linked in a construct of interest or by homologous recombination into the construct of interest or the genome of a host cell). Generally, the nucleic acid fragment contains a polynucleotide encoding a polypeptide of interest and the cassette and restriction sites are designed to facilitate insertion of the cassette into the appropriate reading frame for transcription and translation. The expression cassette can also contain elements that promote the 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: promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

[0155] An "isolated" antibody is one that has been identified, separated, and / or recovered from the components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody and can include enzymes, hormones, and other proteins or nonprotein solutes. In some embodiments, the antibody will be purified to (1) greater than 90% by weight, greater than 95% by weight, or greater than 98% by weight antibody, as determined by the Lowry method, e.g., greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE using Coomassie blue or silver stain under reducing or nonreducing conditions. 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 certain instances, isolated antibodies will be prepared by at least one purification step.

[0156] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are made and paired 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 an antibody-producing cell isolated from a first animal immunized with an antigen is a natural antibody.

[0157] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., murine or rabbit) antibody that contains one or more amino acids (e.g., in the framework region, constant region, or CDRs) that have been replaced with the corresponding-located amino acids from a human antibody. Typically, a humanized antibody produces a reduced immune response in a human host compared to the non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting, veneering or resurfacing, chain shuffling, etc. In certain embodiments, framework substitutions are determined by modeling the interactions of the CDRs and framework residues to identify framework residues important for antigen binding and by 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.

[0158] In certain embodiments, the antibody molecules disclosed herein include a heavy chain comprising a variable heavy chain region 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 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 acids set forth in UniProtKB / Swiss-Prot:P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibody may comprise a mutation, e.g., a substitution for modulating Fc function. For example, the LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody-dependent cell cytotoxicity (ADCC). The numbering of the alternative sequences is based on the EU numbering system. The "EU numbering system" or "EU index" is generally used to refer to the residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The "EU index in the Kabat system" refers to the residue numbering of the human IgG1 EU antibody.

[0159] The term "chimeric antibody" refers to an antibody in which the light and heavy chain genes have been constructed from antibody variable and constant region genes belonging to different species, typically by genetic engineering. For example, the variable fragment of the gene from a murine monoclonal antibody may be ligated to a human constant fragment, e.g., γ1 and γ3. An example of a therapeutic chimeric antibody is a hybrid protein consisting of the variable region or antigen-binding domain from a murine antibody and the constant region or effector domain from a human antibody, although domains from other mammalian species may also be used.

[0160] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless otherwise specified, "polypeptide", "peptide", and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells); immunolabeled proteins, etc. In the context of an antibody, it is apparent that a chain or domain comprises a polypeptide.

[0161] The terms "native amino acid sequence" or "parental amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include modified amino acid residues.

[0162] The terms "amino acid analog", "unnatural amino acid", etc. are used interchangeably and include amino acid-like compounds that are similar in structure and / or overall shape to one or more of the 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 natural amino acids with modified side chains or main chains. Amino acid analogs also include amino acid analogs having the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some cases, amino acid analogs share the main chain structure and / or side chain structure of one or more natural amino acids, differing by one or more modifying groups in the molecule. Such modifications can include, but are not limited to, replacing a relevant atom (e.g., S) with an atom (e.g., N), adding a group (e.g., a methyl or hydroxyl group, etc.) or an atom (e.g., Cl or Br, etc.), deleting a group, replacing a covalent bond (a single bond instead of a double bond, etc.), or a combination thereof. For example, amino acid analogs can include α-hydroxy acids and α-amino acids, etc.

[0163] The term "amino acid side chain" or "side chain of an amino acid", etc. 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 the amino acid side chains of modified amino acids and / or conjugates as described herein, as the context may indicate.

[0164] The term "conjugation" generally refers to a covalent or non-covalent chemical bond, typically covalent, that proximally links a first molecule of interest to a second molecule of interest. In some embodiments, the reagent is selected from a half-life extending moiety, a labeling agent, and a therapeutic agent. For example, to extend the half-life, an antibody of the present disclosure can optionally be modified to provide an improved pharmacokinetic profile (e.g., by polyethylene glycolylation (PEGylation), hyperglycosylation, etc.). Modifications that can increase the serum half-life are of interest.

[0165] The term "carbohydrate", etc. can be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides and polysaccharides. The term sugar can be used to refer to smaller carbohydrates such as monosaccharides, disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (substituted by any suitable substituent), modified (converted to another group using any suitable chemical) or missing (e.g., eliminated or substituted by H). A variety of carbohydrates and carbohydrate derivatives are available and can be applicable to the subject compounds and conjugates.

[0166] As used herein, the term "isolated" is intended to describe a compound of interest in an environment different from that in which it naturally occurs. "Isolated" is intended to include compounds in such samples in which the compound of interest has been substantially enriched and / or in which the compound of interest has been partially or substantially purified.

[0167] As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment and is separated from other components naturally associated therewith by at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or greater than 98%.

[0168] The term "physiological conditions" refers to those conditions that are compatible with living cells, e.g., temperature, pH, salinity, etc. of the predominantly aqueous conditions compatible with living cells.

[0169] "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 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 the converted aldehyde tag (e.g., the reactive aldehyde group) and an "aldehyde-reactive reactive partner" that contains an aldehyde-reactive group and a moiety of interest, and which reacts to form a reaction product of a polypeptide that has the moiety of interest conjugated to the polypeptide through the fGly residue.

[0170] The "N-terminus" refers to the terminal amino acid residue of a polypeptide that has a free amino group, and the amino groups in non-N-terminal amino acid residues generally form part of the polypeptide covalent backbone.

[0171] The "C-terminus" refers to the terminal amino acid residue of a polypeptide that has a free carboxyl group, and the carboxyl groups in non-C-terminal amino acid residues generally form part of the polypeptide covalent backbone.

[0172] The "internal site" used in a polypeptide or polypeptide amino acid sequence refers to a region in the polypeptide that is not at the N-terminus or C-terminus.

[0173] As used herein, the terms "treatment", "treating", etc. refer to obtaining the desired pharmacological and / or physiological effect. For complete or partial prevention of a disease or its symptoms, the effect can be prophylactic, and / or for partial or complete cure of the disease and / or side effects attributable to the disease, the effect can be therapeutic. "Treatment" as used herein includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a subject who may be predisposed to a disease but has not been diagnosed as having the disease from developing the disease; (b) inhibiting the disease, e.g., preventing its progression; and (c) alleviating the disease, e.g., causing regression of the disease.

[0174] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to, murine (rat, mouse), non-human primate, human, canine, feline, ungulate (e.g., horse, cow, sheep, pig, goat), etc.

[0175] A "therapeutically effective amount" or "effective amount" refers to the amount of anti-MUC1 Ab in a subject sufficient to produce an effect for such treatment of a disease when administered to a mammal or other subject for treating the disease. The "therapeutically effective amount" will vary depending on the anti-MUC1 Ab of the subject being treated, the disease and its severity, and the age, weight, etc.

[0176] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as these embodiments will of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0177] Where a range of values is provided, it should be understood that unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the specified range, to one tenth of the unit of the lower limit, is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, subject to any specifically excluded limitations of the range. Where the range includes one or both of the limiting values, ranges excluding one or both of the included limiting values are also included in the present invention.

[0178] 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 those now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications.

[0179] It must 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, reference to "an antibody" includes a plurality of such antibodies, reference to "a CDR" includes one or more CDRs and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may optionally exclude any optional element. Thus, this statement is intended to serve as a basis for the use of exclusive terms such as "only", "solely" or the use of "negative" limitations when reciting claim elements.

[0180] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently confirmed. Detailed Description

[0181] The present disclosure provides conjugates of antibodies specific for MUC1 (e.g., antibody-drug conjugates (ADCs)). The present disclosure also includes methods for producing such conjugates, as well as methods of using them. Each embodiment is described in more detail below. Compositions comprising the ADCs of the present disclosure are also provided, including in some cases pharmaceutical compositions. In certain aspects, methods of using the ADCs are provided, including administering a therapeutically effective amount of the disclosed ADCs to an individual having a cell proliferative disease.

[0182] MUC1 Antibodies and Antibody - Drug Conjugates

[0183] As described above, the present disclosure provides conjugates of antibodies specific for MUC1 (e.g., antibody-drug conjugates (ADCs)). In addition, the present disclosure provides anti-MUC1 antibodies comprising fGly residues.

[0184] Antibody-Drug Conjugates

[0185] The present disclosure provides antibody conjugates specific for MUC1 (e.g., antibody-drug conjugates (ADCs)). "Conjugation" refers to the covalent attachment of a polypeptide (e.g., an antibody) to a moiety of interest (e.g., a drug or an 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 the one or more drugs or active agents are bound to each other through 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 as described herein.

[0186] In certain embodiments, the conjugate is a polypeptide conjugate that comprises 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, such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilizing the polypeptide to a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, where the polypeptide is an antibody, thereby providing an antibody-drug conjugate. For example, the conjugate can be a drug conjugate where the polypeptide is conjugated to one or more drugs or active agents. A variety of drugs and active agents can be used in the conjugate and are described in more detail below.

[0187] One or more drugs or active agents can be conjugated to the polypeptide (e.g., an antibody) at any desired site of the polypeptide. Thus, the present disclosure provides, for example, a polypeptide that is conjugated to a drug or an active agent at a site at or near its C-terminus. Other examples include polypeptides conjugated to a drug or an active agent at a position at or near the N-terminus of the polypeptide. Examples also include polypeptides conjugated to a drug or an active agent at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). When the polypeptide is conjugated to two or more drugs or active agents, the above combinations are also possible.

[0188] In certain embodiments, the conjugates of the present disclosure comprise one or more drugs or active agents conjugated to an amino acid residue of the polypeptide at the α-carbon of the amino acid residue. In other words, the conjugate comprises a polypeptide where the side chain of one or more amino acid residues in the polypeptide has been modified and linked to one or more drugs or active agents (e.g., linked to one or more drugs or active agents through a linker as described herein). For example, the conjugate comprises a polypeptide where the α-carbon of one or more amino acid residues in the polypeptide has been modified and linked to one or more drugs or active agents (e.g., linked to one or more drugs or active agents through a linker as described herein).

[0189] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 or more moieties. These moieties can be conjugated to the 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. The above combinations are also possible, such as a polypeptide conjugated to a first moiety at a first amino acid residue and conjugated 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 a first and a second moiety at a first amino acid residue and a polypeptide conjugated to a third and a fourth moiety at a second amino acid residue, etc.

[0190] One or more amino acid residues of the polypeptide conjugated to one or more moieties of interest can be natural amino acids, unnatural amino acids, or combinations thereof. For example, the conjugate can include one or more drugs or active agents conjugated to a natural amino acid residue of the polypeptide. In other cases, the conjugate can include one or more drugs or active agents conjugated to an unnatural amino acid residue of the polypeptide. As described herein, one or more drugs or active agents can be conjugated to the polypeptide at a single natural or unnatural amino acid residue. One or more natural or unnatural amino acid residues in the polypeptide can be conjugated to one or more moieties described herein. For example, two (or more) amino acid residues (such as, natural or unnatural amino acid residues) in the polypeptide can each be conjugated to one or two moieties, such that multiple sites in the polypeptide are conjugated to the moieties of interest.

[0191] In certain embodiments, a polypeptide (such as, an antibody) and a moiety of interest (such as, a drug or active agent) are conjugated through a conjugating moiety. For example, the polypeptide and the moiety of interest can each be bound to the conjugating moiety (such as, covalently bound), and thus the polypeptide and the moiety of interest are indirectly bound together through the conjugating moiety. In some cases, the conjugating moiety includes a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl compound, or a derivative of a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl compound. For example, a general scheme for conjugating a moiety of interest to a polypeptide through a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugating moiety is shown in the following general reaction scheme. The hydrazino-indolyl and hydrazino-pyrrolo-pyridyl conjugating moieties are also referred to herein as hydrazino-iso-Pictet-Spengler (HIPS) conjugating moieties and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugating moieties, respectively.

[0192] or

[0193]

[0194] In the above reaction scheme, each R comprises a moiety of interest (e.g., a drug or bioactive agent) conjugated to a polypeptide (e.g., conjugated to the polypeptide via a cleavable linker as described herein), where n is an integer from 1 to 4. As shown in the above reaction scheme, a polypeptide comprising a 2-formylglycine residue (fGly) reacts with a drug or bioactive agent that has been modified to comprise a conjugated moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugated moiety) to produce a polypeptide conjugate linked to the conjugated moiety, thereby linking the drug or bioactive agent to the polypeptide via the conjugated moiety.

[0195] As described herein, the 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 bioactive agent. R' and R'' can each independently be any desired substituent, such as, 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. Z can be CR 61 , NR 62 , N, O or S, where R 61 and R 62 are each independently selected from any of the substituents described above for R' and R''.

[0196] As shown by the conjugates and compounds described herein, other hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties are possible. For example, a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety can be linked (e.g., covalently linked) to a linker. Accordingly, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugated moieties linked to a drug or bioactive agent via a linker. Various embodiments of linkers that can couple a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugated moiety to a drug or bioactive agent are described in detail herein. For example, in some cases, as described herein, the linker is a cleavable linker.

[0197] As shown by the conjugates and compounds described herein, additional hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moieties are also possible. For example, a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety can be linked (e.g., covalently linked) to two or more linkers. Accordingly, embodiments of the present disclosure include a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety being linked (e.g., covalently linked) to two or more drugs or active agents via respective linkers. Thus, the conjugates of the present disclosure can comprise two or more linkers, where each linker links a respective drug or active agent to the hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety. Thus, the hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety and two or more linkers can generally be regarded as a “branched linker,” where the hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety is linked to two or more “branches,” where each branch comprises a linker linked to a drug or active agent.

[0198] Combinations of the same or 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) linked 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 linked to a payload (e.g., a drug, active agent, or detectable label), and the second branch of the branched linker can be linked to the same payload (e.g., a drug, active agent, or detectable label) as the first branch.

[0199] In other embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) linked to the branched linker are different payloads (e.g., drugs, active agents, or detectable labels). For example, the first branch of the branched linker can be linked to a first payload (e.g., a first drug, active agent, or detectable label), and the second branch of the branched linker can be linked to a second payload (e.g., a second drug, active agent, or detectable label) different from the first payload (e.g., a first drug, active agent, or detectable label) linked to the first branch.

[0200] 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 result in a polypeptide containing one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide can comprise one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to a moiety of interest (e.g., one or more moieties including a conjugation moiety, such as, for example, a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety as described above). For example, an amino acid of the polypeptide can be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). The reactive aldehyde can be included 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 by the action of formylglycine-generating enzyme (FGE) to contain a 2-formylglycine residue (referred to herein as “fGly”). The fGly residue produced by FGE can also be referred to as “formylglycine”. In other words, the term “aldehyde tag” as used herein refers to an amino acid sequence comprising 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, such as, for example, L(fGly)TPSR (SEQ ID NO:24)). The converted sulfatase motif can be produced from an amino acid sequence comprising an “unconverted” sulfatase motif (e.g., a sulfatase motif in which a cysteine or serine residue has not been converted to fGly by FGE but is capable of being converted, such as the unconverted sulfatase motif having the sequence L(C / S)TPSR). The use of “conversion” 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). U.S. Patent No. 7,985,783 and U.S. Patent No. 8,729,232 describe other aspects of aldehyde tags and their use in site-specific protein modification, the disclosures of each of which are incorporated herein by reference.

[0201] In some cases, to generate a conjugate, a polypeptide containing an fGly residue can be conjugated to a moiety of interest via the reaction of fGly with a compound (e.g., a compound containing a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugation moiety as described above). For example, an fGly - containing polypeptide can be contacted with a drug containing a reactive participant under conditions suitable to provide binding of the drug to the polypeptide. In some cases, the drug containing a reactive participant can include a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugation moiety as described above. For example, a drug or an active agent can be modified to include a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugation moiety. In some cases, the drug or active agent is linked to the hydrazino - indolyl or hydrazino - pyrrolo - pyridyl, for example, covalently linked to the hydrazino - indolyl or hydrazino - pyrrolo - pyridyl via a linker (e.g., a linker described in detail herein).

[0202] In certain embodiments, the conjugates of the present disclosure comprise a polypeptide (e.g., an antibody) having at least one amino acid residue that has been linked to one or more moieties of interest (e.g., a drug or an active agent). To prepare the conjugate, an amino acid residue of the polypeptide can be modified and then coupled to one or more drugs or active agents, which are linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl 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 that has been 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 hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugation moiety as described above to provide a conjugate of the present disclosure, wherein one or more drugs or active agents are conjugated to the polypeptide via the hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugation moiety. As used herein, the term "fGly'" refers to a modified amino acid residue of a polypeptide (e.g., an antibody) conjugated to a moiety of interest (e.g., a drug or an active agent).

[0203] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one amino acid residue linked to a linker as described herein, which linker is in turn linked to one or more drugs or active agents. For example, the conjugate can comprise a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') that is conjugated to one or more moieties of interest (e.g., one or more drugs or active agents) as described above.

[0204] Aspects of the present disclosure include conjugates of formula (I):

[0205]

[0206] wherein

[0207] Z is CR 4or N;

[0208] R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group;

[0209] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, or R 2 and R 3 are optionally joined cyclically to form a 5- or 6-membered heterocyclic group;

[0210] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group;

[0211] L is a linker;

[0212] W 1 is a drug; and

[0213] W 2 is an anti-MUC1 antibody.

[0214] In certain embodiments, Z is CR 4 or N. In certain embodiments, Z is CR 4 . In certain embodiments, Z is N.

[0215] In certain embodiments, R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group.

[0216] In certain embodiments, R 1 is hydrogen. In certain embodiments, R 1 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 1 is methyl. In certain embodiments, R 1 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 1 is alkynyl or substituted alkynyl, such as 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 substituted alkenyl. In certain embodiments, R 1 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 1 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 1 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 1 is heterocyclic or substituted heterocyclic, such as C 3-8 heterocyclic or C 3-8 substituted heterocyclic, such as C 3-6 heterocyclic or C 3-6 substituted heterocyclic, or C 3-5 heterocyclic or C 3-5 substituted heterocyclic.

[0217] In certain embodiments, R 2 and R 3Each 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, or R 2 and R 3 Optionally cyclically linked to form a 5- or 6-membered heterocyclic group.

[0218] In certain embodiments, R 2 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group. In certain embodiments, R 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, such as 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 substituted alkyl. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 alkylamide or substituted alkylamide. In certain embodiments, R 2 is sulfonyl. In certain embodiments, R 2is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 2 is an aryl or a substituted aryl, such as a C 5-8 aryl or a C 5-8 substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl. In certain embodiments, R 2 is a heteroaryl or a substituted heteroaryl, such as a C 5-8 heteroaryl or a C 5-8 substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 2 is a cycloalkyl or a substituted cycloalkyl, such as a C 3-8 cycloalkyl or a C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or a C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or a C 3-5 substituted cycloalkyl. In certain embodiments, R 2 is a heterocyclic group or a substituted heterocyclic group, such as a C 3-6 heterocyclic group or a C 3-6 substituted heterocyclic group, or a C 3-5 heterocyclic group or a C 3-5 substituted heterocyclic group.

[0219] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group. In certain embodiments, R 3 is hydrogen. In certain embodiments, R 3 is alkyl or substituted alkyl, such as a C 1-6 alkyl or a C 1-6 substituted alkyl, or a C 1-4 alkyl or a C 1-4 substituted alkyl, or a C 1-3 alkyl or a C 1-3 substituted alkyl. In certain embodiments, R 3 is methyl. In certain embodiments, R 3 is alkenyl or substituted alkenyl, such as a C 2-6 alkenyl or a C 2-6 substituted alkenyl, or a C 2-4 alkenyl or a C 2-4 substituted alkenyl, or a C 2-3 alkenyl or a C2-3 Substituted alkenyl. In certain embodiments, R 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 carboxylate 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 alkylamide or 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, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 3 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 3 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 3 is heterocyclic or substituted heterocyclic, such as C 3-8 heterocyclic or C 3-8 substituted heterocyclic, such as C 3-6 heterocyclic or C 3-6 substituted heterocyclic, or C 3-5 heterocyclic or C 3-5 substituted heterocyclic.

[0220] In certain embodiments, R 2 and R 3 are optionally ring-linked to form a 5- or 6-membered heterocyclic group. In certain embodiments, R 2 and R 3 are ring-linked to form a 5- or 6-membered heterocyclic group. In certain embodiments, R 2 and R 3A cyclic linkage forms a 5-membered heterocyclic group. In certain embodiments, R 2 and R 3 form a cyclic linkage to form a 6-membered heterocyclic group.

[0221] In certain embodiments, 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0222] The various possibilities for each R 4 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, such as F, Cl, Br, or I. In certain embodiments, R 4 is F. 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, such as 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 substituted alkyl. In certain embodiments, R 4 is methyl. In certain embodiments, R 4 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 4 is alkynyl or substituted alkynyl. In certain embodiments, R 4 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, R4 is an amido or aminoacyl group. In certain embodiments, R 4 is an alkylamide or a substituted alkylamide. In certain embodiments, R 4 is a sulfonyl group. In certain embodiments, R 4 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 4 is an aryl or a substituted aryl, such as a C 5-8 aryl or a C 5-8 substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl (e.g., phenyl or a substituted phenyl). In certain embodiments, R 4 is a heteroaryl or a substituted heteroaryl, such as a C 5-8 heteroaryl or a C 5-8 substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 4 is a cycloalkyl or a substituted cycloalkyl, such as a C 3-8 cycloalkyl or a C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or a C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or a C 3-5 substituted cycloalkyl. In certain embodiments, R 4 is a heterocyclic group or a substituted heterocyclic group, such as a C 3-8 heterocyclic group or a C 3-8 substituted heterocyclic group, such as a C 3-6 heterocyclic group or a C 3-6 substituted heterocyclic group, or a C 3-5 heterocyclic group or a C 3-5 substituted heterocyclic group.

[0223] In certain embodiments, W 1 is a drug. Further description of the drug can be found in the disclosure herein.

[0224] 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, as described herein, the anti-MUC1 antibody is linked to the remainder of the conjugate via an fGly’ residue. Further description of the anti-MUC1 antibody for use in conjugates for a subject can be found in the disclosure herein.

[0225] In certain embodiments, the compounds of formula (I) include a linker, L. This linker can be used to attach a conjugate moiety (e.g., a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety) to one or more target moieties. The linker can be attached (e.g., covalently) to the conjugate moiety at any convenient position (e.g., as described herein). For example, the linker can be modified to include a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety. The hydrazino - indolyl or hydrazino - pyrrolo - pyridyl coupling moiety can be used to conjugate the linker (and the drug) to a polypeptide, such as an anti - MUC1 antibody. For example, the conjugate moiety can be used to conjugate the linker (and the drug) to a modified amino acid residue of the polypeptide, such as the fGly residue of an anti - MUC1 antibody.

[0226] In certain embodiments, L connects the conjugate moiety to W 1 , and thus the conjugate moiety is indirectly attached to W via the linker L 1 . As described above, W 1 is a drug, and thus L connects the conjugate moiety to the drug, e.g., the conjugate moiety is indirectly attached to the drug via the linker L.

[0227] Any suitable linker can be used for the subject conjugates. In certain embodiments, L includes a group selected from: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, amido, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In certain embodiments, L includes an alkyl or substituted alkyl group. In certain embodiments, L includes an alkenyl or substituted alkenyl group. In certain embodiments, L includes an alkynyl or substituted alkynyl group. In certain embodiments, L includes an alkoxy or substituted alkoxy group. In certain embodiments, L includes an amino or substituted amino group. In certain embodiments, L includes a carboxyl or carboxyl ester group. In certain embodiments, L includes an amido group. In certain embodiments, L includes an alkylamide or substituted alkylamide group. In certain embodiments, L includes an aryl or substituted aryl group. In certain embodiments, L includes a heteroaryl or substituted heteroaryl group. In certain embodiments, L includes a cycloalkyl or substituted cycloalkyl group. In certain embodiments, L includes a heterocyclic or substituted heterocyclic group.

[0228] In certain embodiments, L comprises a polymer. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted with an alkyl group at one end), 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 shown by the conjugates and compounds described in more detail below.

[0229] In some embodiments, L is a linker described by the formula:

[0230] -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -,

[0231] wherein L 1 , L 2 , L 3 , L 4 , L 5 and L 6 are each independently linker subunits, and a, b, c, d, e, and f are each independently 0 or 1, where the sum of a, b, c, d, e, and f is 1 to 6.

[0232] 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.

[0233] In certain embodiments, the linker subunit L1 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety (such as, as shown in formula (I) above). In certain embodiments, the linker subunit L 2 , if present, is linked to the drug. In certain embodiments, the linker subunit L 3 , if present, is linked to the said drug. In certain embodiments, the linker subunit L 4 , if present, is linked to the said drug. In certain embodiments, the linker subunit L 5 , if present, is linked to the said drug. In certain embodiments, the linker subunit L 6 , if present, is linked to the said drug.

[0234] Any convenient linker subunit can be used for linker L. Exemplary linker subunits include, but are not limited to, polymer units such as polyethylene glycol, polyethylene, and polyacrylates, amino acid residues, carbohydrate - based polymers or carbohydrate residues and their derivatives, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, each L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 (if present) contains one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, and diamines (such as, a linking group including an alkylenediamine).

[0235] In some embodiments, L 1 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 1 contains polyethylene glycol. In some embodiments, L 1 contains modified polyethylene glycol. In some embodiments, L 1 contains amino acid residues. In some embodiments, L 1 contains an alkyl or substituted alkyl. In some embodiments, L 1 contains an aryl group or a substituted aryl group. In some embodiments, L 1 contains a diamine (such as, a linking group including an alkylenediamine).

[0236] In some embodiments, L 2 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L2 contains polyethylene glycol. In some embodiments, L 2 contains modified polyethylene glycol. In some embodiments, L 2 contains amino acid residues. In some embodiments, L 2 contains an alkyl or substituted alkyl. In some embodiments, L 2 contains an aryl group or a substituted aryl group. In some embodiments, L 2 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0237] In some embodiments, L 3 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 3 contains polyethylene glycol. In some embodiments, L 3 contains modified polyethylene glycol. In some embodiments, L 3 contains amino acid residues. In some embodiments, L 3 contains an alkyl or substituted alkyl. In some embodiments, L 3 contains an aryl group or a substituted aryl group. In some embodiments, L 3 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0238] In some embodiments, L 4 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 contains polyethylene glycol. In some embodiments, L 4 contains modified polyethylene glycol. In some embodiments, L 4 contains amino acid residues. In some embodiments, L 4 contains an alkyl or substituted alkyl. In some embodiments, L 4 contains an aryl group or a substituted aryl group. In some embodiments, L 4 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0239] In some embodiments, L 5 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 contains polyethylene glycol. In some embodiments, L 5 contains modified polyethylene glycol. In some embodiments, L 5contains amino acid residues. In some embodiments, L 5 contains an alkyl or substituted alkyl. In some embodiments, L 5 contains an aryl group or a substituted aryl group. In some embodiments, L 5 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0240] In some embodiments, L 6 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 6 contains polyethylene glycol. In some embodiments, L 6 contains modified polyethylene glycol. In some embodiments, L 6 contains amino acid residues. In some embodiments, L 6 contains an alkyl or substituted alkyl. In some embodiments, L 6 contains an aryl group or a substituted aryl group. In some embodiments, L 6 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0241] In some embodiments, L is a linker containing -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, where:

[0242] -(L 1 ) a - is -(T 1 -V 1 ) a -;

[0243] -(L 2 ) b - is -(T 2 -V 2 ) b -;

[0244] -(L 3 ) c - is -(T 3 -V 3 ) c -;

[0245] -(L4 ) a - is - (T 4 - V 4 ) a -;

[0246] -(L 5 ) b - is - (T 5 - V 5 ) b -; and

[0247] -(L 6 ) c - is - (T 6 - V 6 ) c -;

[0248] wherein T 1 、T 2 、T 3 、T 4 、T 5 and T 6 ,if present, are tether groups;

[0249] V 1 、V 2 、V 3 、V 4 、V 5 and V 6 ,if present, are covalent bonds or linking functional groups; and

[0250] a, b, c, d, e, and f are each independently 0 or 1, where the sum of a, b, c, d, e, and f is 1 to 6.

[0251] As described above, in certain embodiments, L 1 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety (such as, as shown in formula (I) above). Thus, in certain embodiments, T 1 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety (such as, as shown in formula (I) above). In certain embodiments, V 1 is linked to the drug. In certain embodiments, L 2 , if present, is linked to the drug. Thus, in certain embodiments, T 2 , if present, is linked to the drug, or V 2 , if present, is linked to the drug. In certain embodiments, L 3 , if present, is linked to the drug. Thus, in certain embodiments, T 3, if present, is connected to the drug, or V 3 , if present, is linked to the drug. In certain embodiments, L 4 , if present, is linked to the drug. Thus, in certain embodiments, T 4 , if present, is connected to the drug, or V 4 , if present, is linked to the drug. In certain embodiments, L 5 , if present, is linked to the drug. Thus, in certain embodiments, T 5 , if present, is connected to the drug, or V 5 , if present, is linked to the drug. In certain embodiments, L 6 , if present, is linked to the drug. Thus, in certain embodiments, T 6 , if present, is connected to the drug, or V 6 , if present, is connected to the drug.

[0252] About the tether group T 1 , T 2 , T 3 , T 4 , T 5 and T 6 Any suitable tethering group can be used for the subject linker. In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 Each comprises one or more independently selected from covalent bonds, (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w , (PEG) n 、(AA) p -(CR 13 OH) m -, 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide and ester, 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.

[0253] In certain embodiments, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises a (C1-C 12 ) alkyl or a substituted (C1-C 12 ) alkyl. In certain embodiments, the (C1-C 12 ) alkyl is a straight-chain or branched-chain alkyl group comprising from 1 to 12 carbon atoms, such as from 1 to 10 carbon atoms, or from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms, or from 1 to 5 carbon atoms, or from 1 to 4 carbon atoms, or from 1 to 3 carbon atoms. In some cases, the (C1-C 12 ) alkyl can be an alkyl or a substituted alkyl, such as a C1-C 12 alkyl, or a C1-C 10 alkyl, or a C1-C6 alkyl, or a C1-C3 alkyl. In some cases, the (C1-C 12 ) alkyl is a C2 alkyl. For example, the (C1-C 12 ) alkyl can be an alkylene or a substituted alkylene, such as a C1-C 12 alkylene, or a C1-C 10 alkylene, or a C1-C6 alkylene, or a C1-C3 alkylene. In some cases, the (C1-C 12 ) alkyl is a C2 alkylene (e.g., CH2CH2).

[0254] In certain embodiments, the substituted (C1-C 12 ) alkyl is a straight-chain or branched-chain substituted alkyl group comprising from 1 to 12 carbon atoms, such as from 1 to 10 carbon atoms, or from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms, or from 1 to 5 carbon atoms, or from 1 to 4 carbon atoms, or from 1 to 3 carbon atoms. In some cases, the substituted (C1-C 12 ) alkyl can be an alkyl or a substituted alkyl, such as a substituted C1-C 12 alkyl, or a substituted C1-C 10 alkyl, or a substituted C1-C6 alkyl, or a substituted C1-C3 alkyl. In some cases, the substituted (C1-C 12 ) alkyl is a substituted C2 alkyl. For example, the substituted (C1-C 12 ) alkyl can be a substituted alkylene, such as a substituted C1-C 12 alkylene, or a substituted C1-C 10 alkylene, or a substituted C1-C6 alkylene, or a substituted C1-C3 alkylene. In some cases, the substituted (C1-C 12 ) alkyl is a substituted C2 alkylene.

[0255] In certain embodiments, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group or substituted heterocyclic group. In some cases, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises an aryl or substituted aryl. For example, the aryl may be phenyl. In certain cases, the substituted aryl is a substituted phenyl. The substituted phenyl may be substituted with one or more substituents selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group. In some cases, the substituted aryl is a substituted phenyl, wherein the substituent comprises a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0256] In some cases, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises a heteroaryl or substituted heteroaryl. In some cases, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises a cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) comprises a heterocyclic group or substituted heterocyclic group. In some cases, the substituent on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclic group comprises a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0257] In certain embodiments, the tethering group (e.g., T 1 , T 2, T 3 , T 4 , T 5 and / or T 6 ) includes an ethylenediamine (EDA) moiety, such as a tether group containing EDA. In certain embodiments, (EDA) w contains one or more EDA moieties, for example where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12, or from 1 to 6, such as 1, 2, 3, 4, 5, or 6). The linked ethylenediamine (EDA) moieties may optionally be substituted at one or more suitable positions with any suitable substituent, such as, alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety is described by the following structure:

[0258]

[0259] where y is an integer from 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. 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 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 groups of EDA may be ring-linked, such as, to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 groups are alkyl groups, ring-linked to form a piperazine ring. In certain embodiments, y is 1 and the adjacent R 12 groups are selected from hydrogen, alkyl (such as, methyl), and substituted alkyl (such as, lower alkyl-OH, for example ethyl-OH or propyl-OH).

[0260] In certain embodiments, the tether group (such as, T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6) includes a 4 - amino - pyridine (4AP) moiety (also referred to herein as pyridin - 4 - amino, P4A). The 4AP moiety may optionally be substituted at one or more suitable positions with any suitable substituent, such as an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl, or a substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure:

[0261]

[0262] wherein R 12 is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (such as 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group; in certain embodiments, R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxyl - modified polyethylene glycol.

[0263] In certain embodiments, R 12 includes a polyethylene glycol moiety as described by the following formula: (PEG) k , which can be represented by the following structure:

[0264]

[0265] where k is an integer from 1 to 20, such as 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain cases, 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, heterocyclic group, and substituted heterocyclic group. In certain embodiments, R 17 is COOH.

[0266] In certain embodiments, the tethering group (such as T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) includes (PEG)n , wherein (PEG) n is a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG) n is described by the following structure:

[0267]

[0268] wherein n is an integer from 1 to 50, such as from 1 to 40, or from 1 to 30, or from 1 to 20, or from 1 to 12, or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 6. In certain cases, n is 12.

[0269] In certain embodiments, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) includes (AA) p , wherein AA is an amino acid residue. Any suitable amino acid can be utilized. Exemplary amino acids include, but are not limited to, L- and D-amino acids, any of the 20 major α-amino acids and β-alanine that are naturally occurring amino acids, 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, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 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.

[0270] In certain embodiments, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6) includes amino acid analogs. Amino acid analogs include compounds that are similar in structure and / or overall shape to one or more amino acids (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) commonly found in naturally occurring proteins. Amino acid analogs also include natural amino acids with modified side chains or main chains. Amino acid analogs also include amino acid analogs having the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some cases, amino acid analogs share the main chain structure and / or side chain structure of one or more natural amino acids, differing by one or more modifying groups in the molecule. Such modifications can include, but are not limited to, replacing a relevant atom (e.g., S) with an atom (e.g., N), adding a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., Cl or Br, etc.), deleting a group, replacing a covalent bond (single bond instead of double bond, etc.), or a combination thereof. For example, amino acid analogs can include α-hydroxy acids and α-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfopropionic acid, etc.

[0271] In certain embodiments, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) includes a moiety described by the formula -(CR 13 OH) m -, where m is 0 or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 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 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group. In certain embodiments, R 13 is hydrogen. In certain embodiments, R 13 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C1-4 a substituted alkyl group, or C 1-3 an alkyl group or C 1-3 a substituted alkyl group. In certain embodiments, R 13 is an alkenyl group or a substituted alkenyl group, such as C 2-6 an alkenyl group or C 2-6 a substituted alkenyl group, or C 2-4 an alkenyl group or C 2-4 a substituted alkenyl group, or C 2-3 an alkenyl group or C 2-3 a substituted alkenyl group. In certain embodiments, R 13 is an alkynyl group or a substituted alkynyl group. In certain embodiments, R 13 is an alkoxy group or a substituted alkoxy group. In certain embodiments, R 13 is an amino group or a substituted amino group. In certain embodiments, R 13 is a carboxyl group or a carboxyl ester. In certain embodiments, R 13 is an acyl group or an acyloxy group. In certain embodiments, R 13 is an acylamino group or an aminoacyl group. In certain embodiments, R 13 is an alkylamide group or a substituted alkylamide group. In certain embodiments, R 13 is a sulfonyl group. In certain embodiments, R 13 is a thioalkoxy group or a substituted thioalkoxy group. In certain embodiments, R 13 is an aryl group or a substituted aryl group, such as C 5-8 an aryl group or C 5-8 a substituted aryl group, such as a C5 aryl group or a C5 substituted aryl group, or a C6 aryl group or a C6 substituted aryl group. In certain embodiments, R 13 is a heteroaryl group or a substituted heteroaryl group, such as C 5-8 a heteroaryl group or C 5-8 a substituted heteroaryl group, such as a C5 heteroaryl group or a C5 substituted heteroaryl group, or a C6 heteroaryl group or a C6 substituted heteroaryl group. In certain embodiments, R 13 is a cycloalkyl group or a substituted cycloalkyl group, such as C 3-8 a cycloalkyl group or C 3-8 a substituted cycloalkyl group, such as C 3-6 a cycloalkyl group or C 3-6 a substituted cycloalkyl group, or C 3-5 a cycloalkyl group or C 3-5 a substituted cycloalkyl group. In certain embodiments, R 13 is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 a heterocyclic group or C 3-8 a substituted heterocyclic group, such as C 3-6 a heterocyclic group or C 3-6 a substituted heterocyclic group, or C 3-5 a heterocyclic group or C 3-5 a substituted heterocyclic group.

[0272] In certain embodiments, each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .

[0273] In certain embodiments, the tethering group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and / or T 6 ) 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).

[0274] In some embodiments, the tethering group includes an MABO group described by the following structure:

[0275]

[0276] In some embodiments, the tethering group includes an MABC group described by the following structure:

[0277]

[0278] In some embodiments, the tethering group includes a PABO group described by the following structure:

[0279]

[0280] In some embodiments, the tethering group includes a PABC group described by the following structure:

[0281]

[0282] In some embodiments, the tethering group includes a PAB group described by the following structure:

[0283]

[0284] In some embodiments, the tethering group includes a PABA group described by the following structure:

[0285]

[0286] In some embodiments, the tethering group includes a PAP group described by the following structure:

[0287]

[0288] In some embodiments, the tethering group comprises a PHP group described by the following structure:

[0289]

[0290] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0291] 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, such as 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 substituted alkyl. In certain embodiments, R 14 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 alkylamide or substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R14 is an aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 14 is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 14 is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 14 is a heterocyclic group or substituted heterocyclic group, such as C 3-8 heterocyclic group or C 3-8 substituted heterocyclic group, such as C 3-6 heterocyclic group or C 3-6 substituted heterocyclic group, or C 3-5 heterocyclic group or C 3-5 substituted heterocyclic group.

[0292] In some of the above embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the benzene ring may be substituted by one or more additional 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0293] In certain embodiments of linker L, one or more of the tether groups T 1 、T 2 、T 3 、T 4 、T 5 、or T 6 are optionally substituted by a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucosidase, O-GlcNAc, and O-GalNAc.

[0294] In certain embodiments, the above-described 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-described MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the benzene 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, fucosidase, O-GlcNAc, and O-GalNAc.

[0295] For example, in some embodiments, the glycoside or glycoside derivative may be selected from the following structures:

[0296]

[0297] and

[0298] Regarding the linking functional group, V 1 、V 2 、V 3 、V 4 、V 5 and V 6 , any suitable linking functional group may be used in the linker L. Exemplary linking functional groups include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonamido, aminosulfonyl, thio, oxy, phospho, aminophosphate, thiophosphoester, and the like. In some embodiments, V 1 、V 2 、V 3 、V 4 、V 5 and V 6 are each independently selected from a covalent bond, -CO-, -NR 15 -, -NR 15 (CH)2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15SO2- and -P(O)OH-, where 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.

[0299] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0300] 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, such as 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 substituted alkyl. In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 alkylamide or substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15 is an aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 15 is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 15 is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 15 is a heterocyclic group or substituted heterocyclic group, such as C 3-8 heterocyclic group or C 3-8 substituted heterocyclic group, such as C 3-6 heterocyclic group or C 3-6 substituted heterocyclic group, or C 3-5 heterocyclic group or C 3-5 substituted heterocyclic group.

[0301] 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, heterocyclic group, and substituted heterocyclic group. In these embodiments, the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group are as described above for R 15 described.

[0302] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0303] As described above, in some embodiments, L is a group containing -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f -type joint, where 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 from 1 to 6.

[0304] In some embodiments, in joint L:

[0305] T 1 is selected from (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl;

[0306] T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from (C1-C 12 ) alkyl, (C1-C 12 ) substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group, (EDA)w, (PEG)n, (AA)p-(CR13OH)m-, 4-aminopiperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal group, disulfide, hydrazine, and ester; and

[0307] V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 are each independently selected from covalent bond, -CO-, -NR 15 -, -NR 15 (CH)2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2-, and -P(O)OH-, where q is an integer from 1 to 6;

[0308] Wherein:

[0309] (PEG) n is wherein n is an integer between 1 and 30;

[0310] EDA is an ethylenediamine moiety having the following structure:

[0311] wherein y is an integer from 1 to 6 and r is 0 or 1;

[0312] 4-aminopiperidine (4AP) is

[0313] AA is an amino acid residue, wherein p is an integer from 1 to 20; and

[0314] Each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring;

[0315] Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0316] 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, heterocyclic group, and substituted heterocyclic group.

[0317] 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 are selected from the following:

[0318] Wherein:

[0319] T 1 is (C1-C 12 ) alkyl, and V 1 is -CO-;

[0320] T 2 is an amino acid analogue and V 2 is -NH-;

[0321] T 3 is (PEG) n and V 3 is -CO-;

[0322] T 4 is AA and V 4 is absent;

[0323] T 5 is PABC and V 5 is absent; and

[0324] f is 0; or

[0325] wherein:

[0326] T 1 is (C1-C 12 ) alkyl, and V 1 is -CO-;

[0327] T 2 is 4AP and V 2 is -CO-;

[0328] T 3 is (C1-C 12 ) alkyl, and V 3 is -CO-;

[0329] d, e, and f are each 0.

[0330] For example, in certain embodiments, the conjugate of formula (I) has a structure selected from the following:

[0331]

[0332] In certain embodiments, the left side of the linker structure is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugate moiety, and the right side of the linker structure is attached to the drug W 1 , as shown above, for example.

[0333] In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and the drug are linked together by a linker (e.g., L), as described above. In some cases, the linker is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where a cleavable moiety includes one or more bonds that can dissociate under certain conditions, thereby dividing the cleavable linker into two or more separable moieties. For example, a cleavable moiety can include one or more covalent bonds that can dissociate or break under certain conditions to separate the cleavable linker into two or more moieties. Thus, a cleavable linker can be included in an antibody-drug conjugate such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at a desired drug action target site.

[0334] In some cases, the cleavable linker includes 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 in order to separate or release the drug from the antibody at a desired drug action target site. 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 includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. "Hinders cleavage" means that the presence of the uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage of the first cleavable moiety, and thus substantially reduces the number of cleavable linkers or prevents their cleavage. For example, the presence of the uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The presence of the second cleavable moiety hinders cleavage of the first cleavable moiety, thereby substantially reducing the amount of drug released from the antibody or preventing drug release. For example, premature release of the drug from the antibody can be significantly reduced or prevented until the antibody-drug conjugate is at or near a desired drug action target site.

[0335] In some cases, due to the second cleavable moiety hindering the cleavage of the first cleavable moiety, 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 hindrance to the cleavage of the first cleavable moiety, thereby allowing the first cleavable moiety to be cleaved. As described above, cleavage of the first cleavable moiety can cause the cleavable linker to dissociate or separate into two or more parts to release the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety occurs substantially less in the presence of an uncleaved second cleavable moiety. Substantially means that in the presence of an uncleaved second cleavable moiety, cleavage of the first cleavable moiety is about 10% or less, such as in the presence of an uncleaved second cleavable moiety, cleavage of the first cleavable moiety is 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.

[0336] In other words, the second cleavable moiety can protect the first cleavable moiety from being cleaved. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from being cleaved, thereby greatly reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired drug action target. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thereby allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which in turn separates or releases the drug from the antibody at the desired drug action target as described above. In some cases, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety itself 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 a 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 by the enzymatic action of an enzyme as described above. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved by the enzymatic action of an enzyme, such as, but not limited to, a peptide, a glycoside, etc. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired site of action, such as the desired site of action of the drug released from an antibody-drug conjugate. In certain cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in large amounts in other regions, such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety can be controlled such that a large amount of cleavage occurs at the desired site of action, while little or no cleavage occurs 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 to treat cancer, such as for delivering a cancer therapeutic agent to a desired site of action where cancer cells are present. In some cases, an enzyme, such as the protease cathepsin B, can be a biomarker of cancer that is overexpressed in cancer cells. The overexpression and thus the localization of certain enzymes in cancer can be used in the context of an enzymatically cleavable moiety of a cleavable linker included in an antibody-drug conjugate of the present disclosure to specifically release a drug at a desired site of action, such as the site of cancer (and the overexpressed enzyme). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (such as a peptide) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be the protease cathepsin B. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (such as a peptide) that can be cleaved by a protease such as cathepsin B.

[0339] In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide suitable for a cleavable linker and can be cleaved by the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzymatically cleavable moieties include, for example, valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), etc. For example, the above-described first cleavable moiety (such as the cleavable moiety that is prevented from premature cleavage by the second cleavable moiety) can include a peptide. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from being cleaved by a protease (such as cathepsin B), thereby greatly reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired site of action of the drug. In some cases, one of the amino acid residues of the peptide that includes the first cleavable moiety is linked to or includes a substituent, where the substituent includes the second cleavable moiety. In some cases, the second cleavable moiety includes a glycoside.

[0340] In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glyosyl). In some cases, compared to a cleavable linker that does not include a glycoside, a glycoside can promote an increase in the hydrophilicity of the cleavable linker. The glycoside can be any glycoside or glycoside derivative suitable for a cleavable linker, and it 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 includes a peptide and the second cleavable moiety includes a glycoside. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucosidase, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable moiety is glucuronide. In some cases, the second cleavable moiety is galactoside. In some cases, the second cleavable moiety is glucoside. In some cases, the second cleavable moiety is mannoside. In some cases, the second cleavable moiety is fucosidase. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0341] The glycoside can be linked (e.g., by a covalent bond) to the cleavable linker through a glycosidic bond. The glycosidic bond can link the glycoside to the cleavable linker through various types of bonds, such as 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., by enzymatic hydrolysis of the glycosidic bond). The glycoside can be removed or cleaved from the cleavable linker by any suitable enzyme capable of cleaving (hydrolyzing) the glycosidic bond that links the glycoside to the cleavable linker. Examples of enzymes useful for mediating the cleavage (hydrolysis) of the glycosidic bond that links the glycoside to the cleavable linker are glucuronidases, glycosidases, such as galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes can also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that links the glycoside to the cleavable linker. In some cases, the enzyme found to mediate the cleavage (hydrolysis) of the glycosidic bond that links the glycoside to the cleavable linker is located at or near the desired site of drug action of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, present in a cell at or near the desired site of drug action of the antibody-drug conjugate. In certain cases, the enzyme is an enzyme present at or near the target, where there is an enzyme that mediates the cleavage of the first cleavable moiety.

[0342] In certain embodiments, the conjugate of formula (I) has a structure selected from the following:

[0343]

[0344] Any chemical entity, drug, linker, and conjugation moiety set forth in the foregoing description and structures can be applicable to the subject conjugate.

[0345] Additional disclosure related to hydrazino-indolyl and hydrazino-pyrrolo-pyridyl compounds and methods of making conjugates can be found in U.S. Patent No. 9,310,374 and U.S. Patent No. 9,493,413, the disclosures of each of which are incorporated herein by reference. Additional disclosure related to cleavable linkers is present in U.S. Provisional Application No. 63 / 214,525, filed June 24, 2021, the disclosure of which is incorporated herein by reference.

[0346] Aspects of the present disclosure include conjugates of formula (II):

[0347]

[0348] Wherein:

[0349] Z 1 、Z 2 、Z 3 And Z 4 Are each independently selected from CR 24 、N, and C-L B -W 12 Wherein, Z 1 、Z 2 、Z 3 And Z 4 At least one is C-L B -W 12 ;

[0350] R 21 Is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic;

[0351] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic, or R 22 And R 23Optionally, it is cyclically linked to form a 5- or 6-membered heterocyclic group;

[0352] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group;

[0353] L A is the first linker;

[0354] L B is the second linker;

[0355] W 11 is the first drug;

[0356] W 12 is the second drug; and

[0357] W 13 is a polypeptide.

[0358] The substituents related to the conjugate of formula (II) are described in more detail below.

[0359] In certain embodiments, Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 24 , N and C-L B -W 12 , wherein Z 1 , Z 2 , Z 3 and Z 4 at least one is C-L B -W 12 ; in certain embodiments, Z 1 is CR 24 . In certain embodiments, Z 1 is N. In certain embodiments, Z 1 is C-L B -W 12 . In certain embodiments, Z 2 is CR 24 . In certain embodiments, Z 2 is N. In certain embodiments, Z 2 is C-L B -W 12 ; in certain embodiments, Z 3is CR 24 。In certain embodiments, Z 3 is N. In certain embodiments, Z 3 is C-L B -W 12 。In certain embodiments, Z 4 is CR 24 。In certain embodiments, Z 4 is N. In certain embodiments, Z 4 is C-L B -W 12 。

[0360] Various Z 1 、Z 2 、Z 3 and Z 4 combinations are possible. For example, in certain cases, Z 1 is C-L B -W 12 、Z 2 is CR 24 、Z 3 is CR 24 and Z 4 is CR 24 。In certain cases, Z 1 is CR 24 、Z 2 is C-L B -W 12 、Z 3 is CR 24 and Z 4 is CR 24 。In certain cases, Z 1 is CR 24 、Z 2 is CR 24 、Z 3 is C-L B -W 12 and Z 4 is CR 24 。In certain cases, Z 1 is CR 24 、Z 2 is CR 24 、Z 3 is CR 24 and Z 4 is C-L B -W 12 。

[0361] In certain embodiments, R 21Selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, substituted heterocyclic group. In certain embodiments, R 21 is hydrogen. In certain embodiments, R 21 is alkyl or substituted alkyl, such as 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 substituted alkyl. In certain embodiments, R 21 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 21 is alkynyl or substituted alkynyl, such as 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 substituted alkenyl. In certain embodiments, R 21 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 21 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 21 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 21 is heterocyclic group or substituted heterocyclic group, such as C 3-8 heterocyclic group or C 3-8 substituted heterocyclic group, such as C 3-6 heterocyclic group or C 3-6 substituted heterocyclic group, or C3-5 heterocyclic group or C 3-5 substituted heterocyclic group.

[0362] In certain embodiments, 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, or R 22 and R 23 are optionally joined cyclically to form a 5- or 6-membered heterocyclic group.

[0363] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group. In certain embodiments, R 22 is hydrogen. In certain embodiments, R 22 is alkyl or substituted alkyl, such as 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 substituted alkyl. In certain embodiments, R 22 is methyl. In certain embodiments, R 22 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 22is an acylamino or aminoacyl group. In certain embodiments, R 22 is an alkylamide or a substituted alkylamide. In certain embodiments, R 22 is a sulfonyl group. In certain embodiments, R 22 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 22 is an aryl or a substituted aryl, such as a C 5-8 aryl or a C 5-8 substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl. In certain embodiments, R 22 is a heteroaryl or a substituted heteroaryl, such as a C 5-8 heteroaryl or a C 5-8 substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 22 is a cycloalkyl or a substituted cycloalkyl, such as a C 3-8 cycloalkyl or a C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or a C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or a C 3-5 substituted cycloalkyl. In certain embodiments, R 22 is a heterocyclic group or a substituted heterocyclic group, such as a C 3-6 heterocyclic group or a C 3-6 substituted heterocyclic group, or a C 3-5 heterocyclic group or a C 3-5 substituted heterocyclic group.

[0364] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group. In certain embodiments, R 23 is hydrogen. In certain embodiments, R 23 is alkyl or substituted alkyl, such as a C 1-6 alkyl or a C 1-6 substituted alkyl, or a C 1-4 alkyl or a C 1-4 substituted alkyl, or a C 1-3 alkyl or a C 1-3 substituted alkyl. In certain embodiments, R 23 is methyl. In certain embodiments, R 23 is alkenyl or substituted alkenyl, such as a C2-6 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 Substituted alkenyl. In certain embodiments, R 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 23 Is amido or aminoacyl. In certain embodiments, R 23 Is alkylamide or 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, such as C 5-8 Aryl or C 5-8 Substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 23 Is heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or C 5-8 Substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 23 Is cycloalkyl or substituted cycloalkyl, such as C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, such as C 3-6 Cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 Substituted cycloalkyl. In certain embodiments, R 23 Is heterocyclic or substituted heterocyclic, such as C 3-8 Heterocyclic or C 3-8 Substituted heterocyclic, such as C 3-6 Heterocyclic or C 3-6 Substituted heterocyclic, or C 3-5 Heterocyclic or C 3-5 Substituted heterocyclic.

[0365] In certain embodiments, R 22 And R 23 Are both methyl.

[0366] In certain embodiments, R 22 and R 23 are optionally linked cyclically to form a 5- or 6-membered heterocyclic group. In certain embodiments, R 22 and R 23 are linked cyclically to form a 5- or 6-membered heterocyclic group. In certain embodiments, R 22 and R 23 are linked cyclically to form a 5-membered heterocyclic group. In certain embodiments, R 22 and R 23 are linked cyclically to form a 6-membered heterocyclic group.

[0367] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0368] The various possibilities for each R 24 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, such as F, Cl, Br, or I. In certain embodiments, R 24 is F. 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, such as 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 substituted alkyl. In certain embodiments, R 24 is methyl. In certain embodiments, R 24 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R24 is an alkynyl or a substituted alkynyl. In certain embodiments, R 24 is an alkoxy or a substituted alkoxy. In certain embodiments, R 24 is an amino or a substituted amino. In certain embodiments, R 24 is a carboxyl or a carboxyl ester. In certain embodiments, R 24 is an acyl or an acyloxy. In certain embodiments, R 24 is an acylamino or an aminoacyl. In certain embodiments, R 24 is an alkylamide or a substituted alkylamide. In certain embodiments, R 24 is a sulfonyl. In certain embodiments, R 24 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 24 is an aryl or a substituted aryl, such as a C 5-8 aryl or a C 5-8 substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl (e.g., a phenyl or a substituted phenyl). In certain embodiments, R 24 is a heteroaryl or a substituted heteroaryl, such as a C 5-8 heteroaryl or a C 5-8 substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 24 is a cycloalkyl or a substituted cycloalkyl, such as a C 3-8 cycloalkyl or a C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or a C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or a C 3-5 substituted cycloalkyl. In certain embodiments, R 24 is a heterocyclic group or a substituted heterocyclic group, such as a C 3-8 heterocyclic group or a C 3-8 substituted heterocyclic group, such as a C 3-6 heterocyclic group or a C 3-6 substituted heterocyclic group, or a C 3-5 heterocyclic group or a C 3-5 substituted heterocyclic group.

[0369] In certain embodiments, L A is a first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0370] In certain embodiments, L B is a second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0371] In certain embodiments, W 11 is a 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.

[0372] 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.

[0373] In certain embodiments, W 13 is a polypeptide (e.g., an antibody). In certain embodiments, W 13 comprises one or more fGly’ residues as described herein. In certain embodiments, as described herein, the polypeptide is linked to the remainder of the conjugate through an fGly’ residue. Examples of polypeptides and antibodies that can be used in the conjugates of the present disclosure are described in more detail below.

[0374] In certain embodiments, the conjugate of formula (II) comprises a first linker, L A . The first linker, L A , can be used to attach a first moiety of interest (e.g., a first drug or active agent) to a polypeptide (e.g., an antibody) through a conjugation moiety. The first linker, L A , can bind (e.g., covalently bind) to a conjugation moiety (e.g., as described herein). For example, the first linker, L A , can link a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety to a first drug. The hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety can be used to conjugate the first linker, L A , (and thus the first drug) to a polypeptide, such as an antibody.

[0375] For example, as shown in formula (II) above, through the conjugation moiety L A is linked to W 13 , and thus W 13 is indirectly bound to the linker L A through a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety. As described above, W 13 is a polypeptide (such as an antibody), and thus L A is linked to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety, for example, the linker L A is indirectly bound to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moiety.

[0376] Any suitable linker can be used for the first linker L A in the subject conjugates and compounds. In certain embodiments, the first linker, L A, including groups selected from the following: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, amido, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group. In certain embodiments, the first linker L A may include an alkyl or substituted alkyl group. In certain embodiments, the first linker L A may include an alkenyl or substituted alkenyl group. In certain embodiments, the first linker L A may include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker L A may include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L A may include an amino or substituted amino group. In certain embodiments, the first linker L A may include a carboxyl or carboxyl ester. In certain embodiments, the first linker L A may include an amido group. In certain embodiments, the first linker L A may include an alkylamide or substituted alkylamide group. In certain embodiments, the first linker L A may include an aryl or substituted aryl group. In certain embodiments, the first linker L A may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L A may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker L A may include a heterocyclic group or substituted heterocyclic group.

[0377] In certain embodiments, the first linker L A may include a polymer. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, 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, polyvinyl pyrrolidone, 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 shown by the conjugates and compounds described in more detail below.

[0378] In some embodiments, L A is a linker described by the following formula:

[0379] -(L 1 ) a -(L 2 ) b -(L 3) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -,

[0380] wherein L 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.

[0381] In certain embodiments, the sum of a, b, c, d, e, and f is from 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.

[0382] In certain embodiments, the linker subunit L 1 is linked to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugate moiety (such as, as shown in formula (I) above). In certain embodiments, if linker subunit L 2 is present, then it is linked to the first drug or active agent W 11 . In certain embodiments, if linker subunit L 3 is present, then it is linked to the first drug or active agent W 11 . In certain embodiments, if linker subunit L 4 is present, then it is linked to the first drug or active agent W 11 . In certain embodiments, if linker subunit L 5 is present, then it is linked to the first drug or active agent W 11 . In certain embodiments, if linker subunit L6 is then linked to said first drug or active agent W 11 .

[0383] Any suitable linker subunit can be used for the first linker L A . Exemplary linker subunits include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene, and polyacrylates, amino acid residues, carbohydrate-based polymers or carbohydrate residues and their derivatives, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, each L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 (if present) contains one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, and diamines (e.g., linking groups including alkylenediamines).

[0384] In some embodiments, L 1 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 1 contains polyethylene glycol. In some embodiments, L 1 contains modified polyethylene glycol. In some embodiments, L 1 contains amino acid residues. In some embodiments, L 1 contains an alkyl or substituted alkyl. In some embodiments, L 1 contains an aryl group or substituted aryl group. In some embodiments, L 1 contains a diamine (e.g., a linking group including an alkylenediamine).

[0385] In some embodiments, L 2 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 2 contains polyethylene glycol. In some embodiments, L 2 contains modified polyethylene glycol. In some embodiments, L 2 contains amino acid residues. In some embodiments, L 2 contains an alkyl or substituted alkyl. In some embodiments, L 2 contains an aryl group or substituted aryl group. In some embodiments, L 2 contains a diamine (e.g., a linking group including an alkylenediamine).

[0386] In some embodiments, L 3 (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 3 comprises polyethylene glycol. In some embodiments, L 3 comprises modified polyethylene glycol. In some embodiments, L 3 comprises amino acid residues. In some embodiments, L 3 comprises an alkyl or a substituted alkyl. In some embodiments, L 3 comprises an aryl group or a substituted aryl group. In some embodiments, L 3 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0387] In some embodiments, L 4 (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 4 comprises polyethylene glycol. In some embodiments, L 4 comprises modified polyethylene glycol. In some embodiments, L 4 comprises amino acid residues. In some embodiments, L 4 comprises an alkyl or a substituted alkyl. In some embodiments, L 4 comprises an aryl group or a substituted aryl group. In some embodiments, L 4 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0388] In some embodiments, L 5 (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 5 comprises polyethylene glycol. In some embodiments, L 5 comprises modified polyethylene glycol. In some embodiments, L 5 comprises amino acid residues. In some embodiments, L 5 comprises an alkyl or a substituted alkyl. In some embodiments, L 5 comprises an aryl group or a substituted aryl group. In some embodiments, L 5 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0389] In some embodiments, L 6(If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 6 includes polyethylene glycol. In some embodiments, L 6 includes modified polyethylene glycol. In some embodiments, L 6 includes amino acid residues. In some embodiments, L 6 includes an alkyl or substituted alkyl. In some embodiments, L 6 includes an aryl group or a substituted aryl group. In some embodiments, L 6 includes a diamine (e.g., a linking group containing an alkylenediamine).

[0390] In some embodiments, L A is a first linker comprising -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, where:

[0391] -(L 1 ) a - is -(T 1 -V 1 ) a -;

[0392] -(L 2 ) b - is -(T 2 -V 2 ) b -;

[0393] -(L 3 ) c - is -(T 3 -V 3 ) c -;

[0394] -(L 4 ) a - is -(T 4 -V 4 ) a -;

[0395] -(L 5 ) b - is -(T 5 -V5 ) b -; and

[0396] -(L 6 ) c - is -(T 6 -V 6 ) c -;

[0397] wherein T 1 、T 2 、T 3 、T 4 、T 5 and T 6 , if present, are tethering groups;

[0398] V 1 、V 2 、V 3 、V 4 、V 5 and V 6 , if present, are covalent bonds or linking functional groups; and

[0399] a, b, d, e, and f are each independently 0 or 1.

[0400] In certain embodiments, the sum of a, b, c, d, e, and f is from 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.

[0401] As described above, in certain embodiments, L 1 is attached to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety (e.g., as shown in formula (II) above). Thus, in certain embodiments, T 1 is attached to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety (e.g., as shown in formula (II) above). In certain embodiments, V1 is linked to a first drug or active agent. In certain embodiments, if linker subunit L 2 is present, it is linked to the first drug or active agent. Thus, in certain embodiments, T 2 , if present, is linked to the first drug or active agent, or V 2 , if present, is linked to the first drug or active agent. In certain embodiments, if linker subunit L 3 is present, it is linked to the first drug or active agent. Thus, in certain embodiments, T 3 , if present, is linked to the first drug or active agent, or V 3 , if present, is linked to the first drug or active agent. In certain embodiments, if linker subunit L 4 is present, it is linked to the first drug or active agent. Thus, in certain embodiments, T 4 , if present, is linked to the first drug or active agent, or V 4 , if present, is linked to the first drug or active agent. In certain embodiments, if linker subunit L 5 is present, it is linked to the first drug or active agent. Thus, in certain embodiments, T 5 , if present, is linked to the first drug or active agent, or V 5 , if present, is linked to the first drug or active agent. In certain embodiments, if linker subunit L 6 is present, it is linked to the first drug or active agent. Thus, in certain embodiments, T 6 , if present, is linked to the first drug or active agent, or V 6 , if present, is linked to the first drug or active agent.

[0402] In certain embodiments, the conjugate of formula (II) comprises a second linker, L B . The second linker, L B , can be used 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. The second linker, L B , can bind (e.g., covalently bind) to a conjugation moiety (e.g., as described herein). For example, the second linker, L B , can link a hydrazino - indolyl or hydrazino - pyrrolo - pyridinyl conjugation moiety to a second drug. The hydrazino - indolyl or hydrazino - pyrrolo - pyridinyl conjugation moiety can be used to conjugate the second linker, L B , (and thus the second drug) to a polypeptide, such as an antibody.

[0403] For example, as shown in formula (II) above, through the conjugating moiety L B is linked to W 13 , and thus W 13 is indirectly bound to the second linker L through a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety B . As described above, W 13 is a polypeptide (such as an antibody), and thus L B is linked to the polypeptide (antibody) through a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety, for example, the linker L B is indirectly bound to the polypeptide (antibody) through a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety.

[0404] Any suitable linker can be used for the second linker L in the subject conjugate and compound B . In certain embodiments, the second linker, L B , comprises a group selected from: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, amido, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic. 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 or substituted amino group. In certain embodiments, the second linker L B may comprise a carboxyl or carboxyl ester. In certain embodiments, the second linker L B may comprise an amido 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 comprise a heterocyclic or substituted heterocyclic group.

[0405] In certain embodiments, the second linker L Bmay include a polymer. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted with an alkyl group at one end), 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 shown by the conjugates and compounds described in more detail below.

[0406] In some embodiments, L B is a second linker described by the following formula:

[0407] -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m ,

[0408] where L 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.

[0409] In certain embodiments, the sum of g, h, i, j, k, l, and m is from 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.

[0410] In certain embodiments, linker subunit L 7 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugating moiety (such as, as shown in formula (II) above). In certain embodiments, if linker subunit L 8 is present, then it is linked to the second drug or active agent W 12 . In certain embodiments, if linker subunit L 9 is present, then it is linked to the second drug or active agent W 12 . In certain embodiments, if linker subunit L 10 is present, then it is linked to the second drug or active agent W 12 . In certain embodiments, if linker subunit L 11 is present, then it is linked to the second drug or active agent W 12 . In certain embodiments, if linker subunit L 12 is present, then it is linked to the second drug or active agent W 12 . In certain embodiments, if linker subunit L 13 is present, then it is linked to the second drug or active agent W 12 .

[0411] Any suitable linker subunit can be used for the second linker L BThe target linker subunit includes, but is not limited to, polymer units such as polyethylene glycol, polyethylene, and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues and their derivatives, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, each L 7 、L 8 、L 9 、L 10 、L 11 and L 13 (if present) contains one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, and diamines (e.g., a linking group including an alkylenediamine).

[0412] In some embodiments, L 7 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 7 contains polyethylene glycol. In some embodiments, L 7 contains modified polyethylene glycol. In some embodiments, L 7 contains amino acid residues. In some embodiments, L 7 contains an alkyl or a substituted alkyl. In some embodiments, L 7 contains an aryl group or a substituted aryl group. In some embodiments, L 7 contains a diamine (e.g., a linking group including an alkylenediamine).

[0413] In some embodiments, L 8 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 8 contains polyethylene glycol. In some embodiments, L 8 contains modified polyethylene glycol. In some embodiments, L 8 contains amino acid residues. In some embodiments, L 8 contains an alkyl or a substituted alkyl. In some embodiments, L 8 contains an aryl group or a substituted aryl group. In some embodiments, L 8 contains a diamine (e.g., a linking group including an alkylenediamine).

[0414] In some embodiments, L 9(If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 9 comprises polyethylene glycol. In some embodiments, L 9 comprises modified polyethylene glycol. In some embodiments, L 9 comprises amino acid residues. In some embodiments, L 9 comprises an alkyl or a substituted alkyl. In some embodiments, L 9 comprises an aryl group or a substituted aryl group. In some embodiments, L 9 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0415] In some embodiments, L 10 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 10 comprises polyethylene glycol. In some embodiments, L 10 comprises modified polyethylene glycol. In some embodiments, L 10 comprises amino acid residues. In some embodiments, L 10 comprises an alkyl or a substituted alkyl. In some embodiments, L 10 comprises an aryl group or a substituted aryl group. In some embodiments, L 10 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0416] In some embodiments, L 11 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 11 comprises polyethylene glycol. In some embodiments, L 11 comprises modified polyethylene glycol. In some embodiments, L 11 comprises amino acid residues. In some embodiments, L 11 comprises an alkyl or a substituted alkyl. In some embodiments, L 11 comprises an aryl group or a substituted aryl group. In some embodiments, L 11 comprises a diamine (e.g., a linking group comprising an alkylenediamine).

[0417] In some embodiments, L 12 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 12Contains polyethylene glycol. In some embodiments, L 12 Contains modified polyethylene glycol. In some embodiments, L 12 Contains amino acid residues. In some embodiments, L 12 Contains an alkyl or substituted alkyl. In some embodiments, L 12 Contains an aryl group or a substituted aryl group. In some embodiments, L 12 Contains a diamine (e.g., a linking group containing an alkylenediamine).

[0418] In some embodiments, L 13 (If present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 13 Contains polyethylene glycol. In some embodiments, L 13 Contains modified polyethylene glycol. In some embodiments, L 13 Contains amino acid residues. In some embodiments, L 13 Contains an alkyl or substituted alkyl. In some embodiments, L 13 Contains an aryl group or a substituted aryl group. In some embodiments, L 13 Contains a diamine (e.g., a linking group containing an alkylenediamine).

[0419] In some embodiments, L B Is a second linker containing -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m -, where:

[0420] -(L 7 ) g - is -(T 7 -V 7 ) g -;

[0421] -(L 8 ) h - is -(T 8 -V 8 ) h -;

[0422] -(L 9 ) i - is -(T 9 -V 9 ) i -;

[0423] -(L 10 ) j - is -(T 10 -V 10 ) j -;

[0424] -(L 11 ) k - is -(T 11 -V 11 ) k -;

[0425] -(L 12 ) l - is -(T 12 -V 12 ) l -; and

[0426] -(L 13 ) m - is -(T 13 -V 13 ) m -;

[0427] wherein T 7 , T 8 , T 9 , T 10 , T 11 and T 13 , if present, are tether groups;

[0428] V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 , if present, are covalent bonds or linking functional groups; and

[0429] g, h, i, j, k, l, and m are each independently 0 or 1.

[0430] In certain embodiments, the sum of g, h, i, j, k, l, and m is from 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, each of g, h, i, j, k, l, and m is 1. In certain embodiments, each of g, h, i, j, k, and l is 1, and m is 0. In certain embodiments, each of g, h, i, j, and k is 1, and each of l and m is 0. In certain embodiments, each of g, h, i, and j is 1, and each of k, l, and m is 0. In certain embodiments, each of g, h, and i is 1, and each of j, k, l, and m is 0. In certain embodiments, each of g and h is 1, and each of i, j, k, l, and m is 0. In certain embodiments, g is 1, and each of h, i, j, k, l, and m is 0. In certain embodiments, each of g, h, i, j, k, l, and m is 0.

[0431] As described above, in certain embodiments, L 7 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety (e.g., as shown in formula (II) above). Thus, in certain embodiments, T 7 is linked to a hydrazino - indolyl or hydrazino - pyrrolo - pyridyl conjugate moiety (e.g., as shown in formula (II) above). In certain embodiments, V 7 is linked to a second drug or active agent. In certain embodiments, if there is a linker subunit L 8 , it is linked to the second drug or active agent. Thus, in certain embodiments, T 8 , if present, is linked to the second drug or active agent, or V 8 , if present, is linked to the second drug or active agent. In certain embodiments, if there is a linker subunit L 9 , it is linked to the second drug or active agent. Thus, in certain embodiments, T 9 , if present, is linked to the second drug or active agent, or V 9 , if present, is linked to the second drug or active agent. In certain embodiments, if there is a linker subunit L 10 , it is linked to the second drug or active agent. Thus, in certain embodiments, T10 , if present, is connected to the second drug or active agent, or V10 4 , if present, is connected to the second drug or active agent. In certain embodiments, if the linker subunit L 11 is present, it is connected to the second drug or active agent. Thus, in certain embodiments, T 11 , if present, is connected to the second drug or active agent, or V 11 , if present, is connected to the second drug or active agent. In certain embodiments, if the linker subunit L 12 is present, it is connected to the second drug or active agent. Thus, in certain embodiments, T 12 , if present, is connected to the second drug or active agent, or V 12 , if present, is connected to the second drug or active agent. In certain embodiments, if the linker subunit L 13 is present, it is connected to the second drug or active agent. Thus, in certain embodiments, T 13 , if present, is connected to the second drug or active agent, or V 13 , if present, is connected to the second drug or active agent.

[0432] Regarding the tethering 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 T 13 , for the subject linker, any suitable tethering group can be used. In some embodiments, 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 T 13 each comprise one or more independent groups selected from covalent bonds, (C1-C 12 ) alkyl, substituted (C1-C 12alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (EDA) w , (PEG) n , (AA) p -(CR 13 OH) m -, 4-aminopiperidine (4AP), meta-aminobenzyloxy (MABO), meta-aminobenzyloxycarbonyl (MABC), para-aminobenzyloxy (PABO), para-aminobenzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-aminobenzylamine (PABA), para-aminophenyl (PAP), para-hydroxyphenyl (PHP), acetal group, hydrazine, disulfide and ester, 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.

[0433] In certain embodiments, 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 ) comprises a (C1-C 12 ) alkyl or a substituted (C1-C 12 ) alkyl. In certain embodiments, the (C1-C 12 ) alkyl is a straight-chain or branched-chain alkyl group that comprises 1 to 12 carbon atoms, such as 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 cases, the (C1-C 12 ) alkyl can be an alkyl or a substituted alkyl, such as a C1-C 12 alkyl, or a C1-C 10 alkyl, or a C1-C6 alkyl, or a C1-C3 alkyl. In some cases, the (C1-C 12 ) alkyl is a C2-alkyl. For example, the (C1-C 12 ) alkyl can be an alkylene or a substituted alkylene, such as a C1-C 12 alkylene, or a C1-C 10 alkylene, or a C1-C6 alkylene, or a C1-C3 alkylene. In some cases, the (C1-C 12) The alkyl group is a C1-alkylene group (e.g., CH2). In some cases, (C1-C 12 ) The alkyl group is a C2-alkylene group (e.g., CH2CH2). In some cases, (C1-C 12 ) The alkyl group is a C3-alkylene group (e.g., CH2CH2CH2).

[0434] In certain embodiments, the substituted (C1-C 12 ) alkyl group is a straight-chain or branched-chain substituted alkyl group, which includes 1 to 12 carbon atoms, such as 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 cases, the substituted (C1-C 12 ) alkyl group can be an alkyl group or a substituted alkyl group, such as a substituted C1-C 12 alkyl group, or a substituted C1-C 10 alkyl group, or a substituted C1-C6 alkyl group, or a substituted C1-C3 alkyl group. In some cases, the substituted (C1-C 12 ) alkyl group is a substituted C2-alkyl group. For example, the substituted (C1-C 12 ) alkyl group can be a substituted alkylene group, such as a substituted C1-C 12 alkylene group, or a substituted C1-C 10 alkylene group, or a substituted C1-C6 alkylene group, or a substituted C1-C3 alkylene group. In some cases, the (C1-C 12 ) alkyl group is a substituted C1-alkylene group (e.g., a C1-alkylene group substituted with -SO3H). In some cases, the substituted (C1-C 12 ) alkyl group is a substituted C2-alkyl group. In some cases, the substituted (C1-C 12 ) alkyl group is a substituted C3-alkyl group. For example, the substituted (C1-C 12 ) alkyl group can include a C1-C k alkylene group (such as a C3-alkylene group or a C5-alkylene group) substituted with a group as described herein (PEG) k , such as -CONH(PEG) k , for example -CONH(PEG)3 or -CONH(PEG)5; or -NHCO(PEG) 12 , for example -NHCO(PEG)7), or can include a C1-C 12 alkylene group (such as a C3-alkylene group) substituted with a -CONHCH2CH2SO3H group, or can include a C1-C 12 alkylene group (such as a C5-alkylene group) substituted with a -NHCOCH2SO3H group.

[0435] In certain embodiments, the tether group (such as T 1 、T2 , 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, heterocyclic or substituted heterocyclic. In certain embodiments, 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 aryl or substituted aryl. For example, the aryl can be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl can be substituted with one or more substituents selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic. In some cases, the substituted aryl is a substituted phenyl, wherein the substituent includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety such as a glycoside or a glycoside derivative).

[0436] In certain embodiments, 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 certain embodiments, 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 a cycloalkyl or a substituted cycloalkyl. In certain embodiments, 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 a heterocyclic group or a substituted heterocyclic group. In some cases, the substitution on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclic group includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety such as a glycoside or a glycoside derivative).

[0437] In certain embodiments, 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 an ethylenediamine (EDA) moiety, such as a tether group comprising EDA. In certain embodiments, the (EDA) w comprises one or more EDA moieties, for example where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6). The linked ethylenediamine (EDA) moieties may optionally be substituted at one or more suitable positions with any suitable substituent, such as, an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the EDA moiety is described by the following structure:

[0438]

[0439] where y is an integer from 1 to 6, or is 0 or 1, and each R 12Independently 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group. 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 is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In certain embodiments, any two adjacent R of EDA 12 groups may be ring-linked, such as to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 groups are alkyl groups, which are ring-linked to form a piperazine ring. In certain embodiments, y is 1 and the adjacent R 12 groups are selected from hydrogen, alkyl (such as methyl) and substituted alkyl (such as lower alkyl-OH, for example ethyl-OH or propyl-OH).

[0440] In certain embodiments, the tether group (such as, 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 a 4-aminopyridine (4AP) moiety (also referred to herein as pyridine-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more suitable positions with any suitable substituent, such as alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl or substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure:

[0441]

[0442] wherein R 12Selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties (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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic; in certain embodiments, R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxyl-modified polyethylene glycol.

[0443] In certain embodiments, R 12 includes a polyethylene glycol moiety as described by the following formula: (PEG) k , which can be represented by the following structure:

[0444]

[0445] where k is an integer from 1 to 20, such as 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain cases, 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, heterocyclic and substituted heterocyclic. In certain embodiments, R 17 is COOH. In certain embodiments, R 17 is OH. In certain embodiments, R 17 is OCH3.

[0446] In certain embodiments, the tethering 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 (PEG) n , where (PEG) nis a polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG)n is described by the following structure:

[0447]

[0448] where n is an integer from 1 to 50, such as from 1 to 40, or from 1 to 30, or from 1 to 20, or from 1 to 12, or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 6. In certain cases, n is 12.

[0449] In certain embodiments, 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 (AA) p , where AA is an amino acid residue. Any suitable amino acid can be utilized. Exemplary amino acids include, but are not limited to, L- and D-amino acids; naturally occurring amino acids, such as any of the 20 major α-amino acids and β-alanine; 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, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12, or from 1 to 6, such as 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.

[0450] In certain embodiments, 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 amino acid analogs. Amino acid analogs include compounds that are similar in structure and / or overall shape to one or more amino acids (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) commonly found in naturally occurring proteins. Amino acid analogs also include natural amino acids with modified side chains or main chains. Amino acid analogs also include amino acid analogs having the same stereochemistry as naturally occurring D- and L-amino acid analogs. In some cases, amino acid analogs share the main chain structure and / or side chain structure of one or more natural amino acids, differing by one or more modifying groups in the molecule. Such modifications can include, but are not limited to, replacing a relevant atom (e.g., S) with an atom (e.g., N), adding a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., Cl or Br, etc.), deleting a group, replacing a covalent bond (single bond instead of double bond, etc.), or a combination thereof. For example, amino acid analogs can include α-hydroxy acids and α-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfoalanine, etc.

[0451] In certain embodiments, 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 the moiety described by the formula -(CR 13 OH) x -, where x is 0 or x is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In certain embodiments, R13 is hydrogen. In certain embodiments, R 13 is alkyl or substituted alkyl, such as 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 substituted alkyl. In certain embodiments, R 13 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 carboxylate 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 alkylamide or 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, such as C 5-8 aryl or C 5-8 substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 13 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 13 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl. In certain embodiments, R 13 is heterocyclic or substituted heterocyclic, such as C 3-8Heterocyclic group or C 3-8 Substituted heterocyclic group, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic group, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic group.

[0452] In certain embodiments, each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .

[0453] In certain embodiments, 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 an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0454] In certain embodiments, 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 meta - amino - benzyloxy (MABO), meta - amino - benzyloxycarbonyl (MABC), para - amino - benzyloxy (PABO), para - amino - benzyloxycarbonyl (PABC), para - aminobenzyl (PAB), para - amino - benzamido (PABA), para - amino - phenyl (PAP), or para - hydroxy - phenyl (PHP).

[0455] In some embodiments, the tether group includes an MABO group described by the following structure:

[0456]

[0457] In some embodiments, the tethering group includes an MABC group described by the following structure:

[0458]

[0459] In some embodiments, the tethering group includes a PABO group described by the following structure:

[0460]

[0461] In some embodiments, the tethering group includes a PABC group described by the following structure:

[0462]

[0463] In some embodiments, the tethering group includes a PAB group described by the following structure:

[0464]

[0465] In some embodiments, the tethering group includes a PABA group described by the following structure:

[0466]

[0467] In some embodiments, the tethering group includes a PAP group described by the following structure:

[0468]

[0469] In some embodiments, the tethering group includes a PHP group described by the following structure:

[0470]

[0471] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group;

[0472] 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, such as C 1-6 alkyl or C1-6 a substituted alkyl, or C 1-4 an alkyl or C 1-4 a substituted alkyl, or C 1-3 an alkyl or C 1-3 a substituted alkyl. In certain embodiments, R 14 is an alkenyl or a substituted alkenyl, such as C 2-6 an alkenyl or C 2-6 a substituted alkenyl, or C 2-4 an alkenyl or C 2-4 a substituted alkenyl, or C 2-3 an alkenyl or C 2-3 a substituted alkenyl. In certain embodiments, R 14 is an alkynyl or a substituted alkynyl. In certain embodiments, R 14 is an alkoxy or a substituted alkoxy. In certain embodiments, R 14 is an amino or a substituted amino. In certain embodiments, R 14 is a carboxyl or a carboxyl ester. In certain embodiments, R 14 is an acyl or an acyloxy. In certain embodiments, R 14 is an acylamino or an aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is a sulfonyl. In certain embodiments, R 14 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 14 is an aryl or a substituted aryl, such as C 5-8 an aryl or C 5-8 a substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl. In certain embodiments, R 14 is a heteroaryl or a substituted heteroaryl, such as C 5-8 a heteroaryl or C 5-8 a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 14 is a cycloalkyl or a substituted cycloalkyl, such as C 3-8 a cycloalkyl or C 3-8 a substituted cycloalkyl, such as C 3-6 a cycloalkyl or C 3-6 a substituted cycloalkyl, or C 3-5 a cycloalkyl or C 3-5 a substituted cycloalkyl. In certain embodiments, R 14 is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 a heterocyclic group or C 3-8 a substituted heterocyclic group, such as C 3-6 a heterocyclic group or C 3-6 a substituted heterocyclic group, or C3-5 heterocyclic group or C 3-5 substituted heterocyclic group.

[0473] In some embodiments of the above-described MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the benzene ring may be substituted with one or more additional 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0474] 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 is / are each optionally substituted with a glycoside or glycoside derivative. For example, in some cases, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside. In some cases, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucosidase, O-GlcNAc, and O-GalNAc.

[0475] In certain embodiments, the 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 MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures described above, the benzene 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, fucosidase, O-GlcNAc, and O-GalNAc.

[0476] For example, in some embodiments, the glycoside or glycoside derivative may be selected from the following structures:

[0477]

[0478] and

[0479] Regarding the 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 , any suitable linking functional group may be used in the subject linker. Exemplary linking functional groups include, but are not limited to, amino, carbonyl, amino, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonamido, aminosulfonyl, thio, oxy, dioxophosphoryl, aminophosphonate, thiophosphonate, and the like. In some embodiments, 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 are each independently selected from a covalent bond, -CO-, -NR 15 -, -NR 15 (CH)2) q -, -NR 15 (C6H4)-, -CONR15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, where 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.

[0480] 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group.

[0481] 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, such as 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 substituted alkyl. In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as 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 substituted alkenyl. In certain embodiments, R 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 15is an amido or aminoacyl group. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is a sulfonyl group. In certain embodiments, R 15 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 15 is an aryl or a substituted aryl, such as a C 5-8 aryl or a C 5-8 substituted aryl, such as a C5 aryl or a C5 substituted aryl, or a C6 aryl or a C6 substituted aryl. In certain embodiments, R 15 is a heteroaryl or a substituted heteroaryl, such as a C 5-8 heteroaryl or a C 5-8 substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. In certain embodiments, R 15 is a cycloalkyl or a substituted cycloalkyl, such as a C 3-8 cycloalkyl or a C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or a C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or a C 3-5 substituted cycloalkyl. In certain embodiments, R 15 is a heterocyclic group or a substituted heterocyclic group, such as a C 3-8 heterocyclic group or a C 3-8 substituted heterocyclic group, such as a C 3-6 heterocyclic group or a C 3-6 substituted heterocyclic group, or a C 3-5 heterocyclic group or a C 3-5 substituted heterocyclic group.

[0482] 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, heterocyclic group, and substituted heterocyclic group. In these embodiments, the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group are as described above for R 15 .

[0483] As described above, in some embodiments, L A is a group containing -(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 - the first linker, where a, b, c, d, e, and f are each independently 0 or 1.

[0484] In some embodiments, in the first linker L A :

[0485] T 1 is selected from (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl;

[0486] T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-aminopiperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal group, disulfide, hydrazine, and ester; and

[0487] V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are each independently selected from covalent bond, -CO-, -NR 15 -, -NR 15 (CH)2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15 -, -NR 15 SO2- and -P(O)OH-, where q is an integer from 1 to 6;

[0488] Where:

[0489] (PEG) n is where n is an integer between 1 and 30;

[0490] EDA is an ethylenediamine moiety having the following structure:

[0491] where y is an integer from 1 to 6 and r is 0 or 1;

[0492] 4-aminopiperidine (4AP) is

[0493] AA is an amino acid residue, where p is an integer from 1 to 20; and

[0494] Each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, where any two adjacent R 12 groups can be cyclically linked to form a piperazinyl ring;

[0495] Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0496] 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, heterocyclic group, and substituted heterocyclic group.

[0497] 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 are selected from the following:

[0498] Where:

[0499] T 1 is (C1-C 12 ) alkyl, and V 1 is -CONH-;

[0500] T 2 is a substituted (C1-C 12 ) alkyl, and V 2 is -CO-;

[0501] T 3 is AA and V 3 is absent;

[0502] T 4 is PABC and V 4 is absent; and

[0503] e and f are each 0.

[0504] In certain embodiments, the left side of the linker structure of the first linker L A is linked to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, and the right side of the linker structure of the first linker L A is linked to a first drug or active agent.

[0505] As described above, in some embodiments, L B is a second linker comprising -(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.

[0506] In some embodiments, in the second linker L B :

[0507] T 7 is selected from (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl;

[0508] T 8 、T 9 、T 10 、T 11 、T12 and T 13 each independently selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x , 4-aminopiperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal group, disulfide, hydrazine and ester; and

[0509] V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 each independently selected from a covalent bond, -CO-, -NR 15 -, -NR 15 (CH)2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, where q is an integer from 1 to 6;

[0510] where:

[0511] (PEG) n is where n is an integer between 1 and 30;

[0512] EDA is an ethylenediamine moiety having the following structure:

[0513] where y is an integer from 1 to 6 and r is 0 or 1;

[0514] 4-aminopiperidine (4AP) is

[0515] AA is an amino acid residue, where p is an integer from 1 to 20; and

[0516] each R 12Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring;

[0517] Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0518] 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, heterocyclic group, and substituted heterocyclic group.

[0519] Any suitable tethering group can be used for T 7 、T 8 、T 9 、T 10 、T 11 、T 12 and T 13 。For example, any one of the above tethering groups related to T 1 、T 2 、T 3 、T 4 、T 5 and T 6 can be used for the tethering group T 7 、T 8 、T 9 、T 10 、T 11 、T 12 and T 13 。

[0520] Any suitable linking functional group can be used for V 7 、V 8 、V 9 、V 10 、V 11 、V 12 and V 13 。For example, any linking functional group related to V 1 、V 2 、V 3 、V 4 、V 5 and V 6 can be used for the linking functional group V 7 、V 8 、V 9 、V 10 、V 11 、V 12 and V 13 。

[0521] In certain embodiments, each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .

[0522] 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, heterocyclic group, and substituted heterocyclic group. In these embodiments, the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group, and substituted heterocyclic group are as described above for R 15 . In these embodiments, the various possible substituents for R 15 are as described above.

[0523] In certain embodiments of the second linker L B , one or more of the tether groups T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , or T 13 are optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucosidase, O-GlcNAc, and O-GalNAc.

[0524] In certain embodiments of the second linker L B , one or more additional groups selected from glycosides and glycoside derivatives may be substituted on the above-described MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures. For example, in some embodiments of the above-described MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures, the benzene 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, fucosidase, O-GlcNAc, and O-GalNAc.

[0525] In certain embodiments, T 7 , T 8 , T 9 , T 10 , T11 , T 12 and T 13 and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 are selected from the following:

[0526] wherein:

[0527] T 7 is absent and V 7 is -NHCO-;

[0528] T 8 is (C1-C 12 ) alkyl, and V 8 is -CONH-;

[0529] T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-;

[0530] T 10 is AA and V 10 is absent;

[0531] T 11 is PABC and V 11 is absent; and

[0532] l and m are each 0.

[0533] In certain embodiments, the second linker L B is attached on the left side of the above linker structure to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugate moiety, and the second linker L B is attached on the right side of the above linker structure to a second drug or active agent.

[0534] In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and the drug are linked together 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 moiety includes one or more bonds that can dissociate under certain conditions, thereby splitting the cleavable linker into two or more separable moieties. For example, the cleavable moiety can include one or more covalent bonds that can dissociate or break under certain conditions to split the cleavable linker into two or more moieties. Thus, the linker contained in an antibody-drug conjugate can be a cleavable linker such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at the desired drug action target site.

[0535] In some cases, the cleavable linker includes two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of two cleavable moieties is required to separate or release the drug from the antibody at the desired drug action target site. 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 includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. By "hindering cleavage" is meant that the presence of the uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage of the first cleavable moiety, thereby substantially reducing the number of cleavable linkers or preventing cleavage of the cleavable linker. For example, the presence of the uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The presence of the second cleavable moiety hinders cleavage of the first cleavable moiety, thereby substantially reducing the amount of drug released from the antibody or preventing drug release from the antibody. For example, premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired drug action target site.

[0536] In some cases, due to the second cleavable moiety hindering the cleavage of the first cleavable moiety, the cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. The cleavage of the second cleavable moiety can reduce or eliminate the hindrance to the cleavage of the first cleavable moiety, thus allowing the first cleavable moiety to be cleaved. The cleavage of the first cleavable moiety can cause the cleavable linker to dissociate or separate into two or more moieties as described above to release the drug from the antibody-drug conjugate. In some cases, the cleavage of the first cleavable moiety occurs substantially in the absence of the uncleaved second cleavable moiety. By substantially it means that in the presence of the uncleaved second cleavable moiety, about 10% or less of the first cleavable moiety is cleaved. For example, in the presence of the uncleaved second cleavable moiety, 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 of the first cleavable moiety is cleaved.

[0537] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of the uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired drug action target. Thus, the cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), allowing the first cleavable moiety to be cleaved, which results in the cleavage of the cleavable linker and thus the separation or release of the drug from the antibody at the desired drug action target as described above. In certain cases, the cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but the cleavage of the second cleavable moiety itself does not result in the cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0538] The cleavable moieties included in the cleavable linker can each 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, such as but not limited to esters, peptides, glycosides, etc. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired site of action, such as the desired drug site of action to be released from the antibody-drug conjugate. In some cases, the amount of the enzyme that cleaves the enzymatically cleavable moiety present in other regions (e.g., in whole blood, plasma, or serum) is not significant. Thus, the cleavage of the enzymatically cleavable moiety can be controlled such that substantially all cleavage occurs at the desired site of action and not significantly in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0539] For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, such as for delivering a cancer therapeutic agent to a desired site of action where cancer cells are present. In certain cases, an enzyme (e.g., an esterase that cleaves an ester bond or a glycosidase that cleaves a glycosidic bond) can be a biomarker for cancer that is overexpressed in cancer cells. The overexpression and thus the localization of certain enzymes in cancer can be used in the context of including an enzymatically cleavable moiety in the cleavable linker of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (i.e., the site of cancer (and the overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or a glycoside) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be an esterase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase. In some cases, the enzyme can be a glycosidase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or a glycoside derivative) that can be cleaved by a glycosidase.

[0540] In certain embodiments, the enzymatically cleavable moiety is an ester bond. For example, the first cleavable moiety described above (i.e., the cleavable moiety that is protected from premature cleavage by the second cleavable moiety) can include an ester. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from being cleaved by an esterase and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired drug site of action. In some cases, a portion of the linker adjacent to the first cleavable moiety is linked to or includes a substituent, where the substituent includes the second cleavable moiety. In some cases, the second cleavable moiety includes a glycoside or a glycoside derivative.

[0541] In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycan) or a glycoside derivative. In some cases, compared with a cleavable linker that does not contain a glycoside or a glycoside derivative, a glycoside or a glycoside derivative can promote an increase in the hydrophilicity of the cleavable linker. The glycoside or glycoside derivative can be any glycoside or glycoside derivative suitable for use in a cleavable linker and 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 a glycoside derivative. For example, in some embodiments, the first cleavable moiety comprises an ester and the second cleavable moiety comprises a glycoside or a glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucosidase, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable moiety is glucuronide. In some cases, the second cleavable moiety is galactoside. In some cases, the second cleavable moiety is glucoside. In some cases, the second cleavable moiety is mannoside. In some cases, the second cleavable moiety is fucosidase. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0542] A glycoside or glycoside derivative can be linked (covalently bonded) to a cleavable linker via a glycosidic bond. The glycosidic bond can link the glycoside or glycoside derivative to the cleavable linker via various types of bonds (such as, 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., by enzymatic-mediated hydrolysis of the glycosidic bond). The glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any suitable enzyme that is capable of cleaving (hydrolyzing) the glycosidic bond that links the glycoside or glycoside derivative to the cleavable linker. Examples of enzymes that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that links the glycoside or glycoside derivative to the cleavable linker are glycosidases, such as glucuronidase, galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes can also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that links the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that links the glycoside or glycoside derivative to the cleavable linker is present at or near the desired site of action of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, present in cells at or near the desired site of action of the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme present at or near the target site where there is an enzyme that mediates the cleavage of the first cleavable moiety.

[0543] Examples of conjugates according to the present disclosure include, but are not limited to, the following structures:

[0544]

[0545] Any of the chemical entities, linkers, and conjugation moieties shown in the above structures can be adapted for use with the subject compounds and conjugates.

[0546] Additional disclosure regarding hydrazino-indolyl and hydrazino-pyrrolo-pyridyl compounds and methods for producing 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.

[0547] Anti-MUC1 antibody

[0548] As described above, the subject conjugate can comprise an anti-MUC1 antibody as 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 their standard name, their standard three-letter abbreviation, and / or their standard one-letter abbreviation, e.g.: 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.

[0549] According to some embodiments, the antibodies of the present disclosure specifically bind MUC1 and comprise:

[0550] A variable heavy chain (VH) chain comprising heavy chain CDRs 1-3 (HCDRs 1-3) of a VH chain having the following sequences:

[0551] EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRP GKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSS LRSEDTAVYYCARGLRYALDYWGQGTLVTVSS (SEQ ID NO:1); and

[0552] A variable light chain (VL) chain comprising light chain CDRs 1-3 (LCDRs 1-3) of a VL chain having the following sequences:

[0553] EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQA PRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCH QYAWSPPTFGQGTKLEIK (SEQ ID NO:2);

[0554] EIVLTQSPATLSLSPGERATLSCRASSSVGSSNLYWYQQKPGQ APRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYC HQYRWSPPTFGQGTKLEIK (SEQ ID NO:3); or

[0555] EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQA PRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQ YSWSPPTFGQGTKLEIK (SEQ ID NO:4).

[0556] HCDR 1-3 and LCDR 1-3 may be defined as per the Chothia, Kabat or IMT nomenclature. The HCDR 1-3 of the anti-MUC1 antibodies disclosed herein as defined according to the listed nomenclatures may be as follows:

[0557] Table 2:

[0558]

[0559] The LCDR 1-3 of the anti-MUC1 antibodies disclosed herein may be as listed in Tables 3-5 as defined according to the nomenclature.

[0560] Table 3

[0561]

[0562] Table 4

[0563]

[0564] Table 5

[0565]

[0566] In certain embodiments, the VH chain of the anti-MUC1 antibody comprises the HCDR 1-3 described herein, and the VL chain of the anti-MUC1 antibody comprises LCDR 1-3, wherein

[0567] LCDR1 comprises the amino acid sequence RASSSVG / SSSYLY (SEQ ID NO:41);

[0568] LCDR2 comprises the amino acid sequence G / RT / SS / TN / KLAS (SEQ ID NO:42);

[0569] LCDR3 comprises the amino acid sequence HQYA / R / SWSPPT (SEQ ID NO:43), according to Kabat definition.

[0570] In certain embodiments, the VH chain of the anti-MUC1 antibody comprises HCDR 1-3 as described herein and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, any amino acid differences between the VH chain of the anti-MUC1 antibody of the present disclosure and SEQ ID NO:1 may be limited to regions outside the CDRs, such as in one or more framework regions (FRs), such as FR1, FR2, FR3, and / or FR4.

[0571] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR 1-3 as shown in Table 3 herein and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:2.

[0572] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR 1-3 as shown in Table 4 herein and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:3.

[0573] In certain embodiments, the VL chain of the anti-MUC1 antibody comprises LCDR 1-3 as shown in Table 5 herein and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:4.

[0574] In certain embodiments, any amino acid differences between the VL chain of the anti-MUC1 antibody of the present disclosure and SEQ ID NO:2, 3, and 4 may be limited to regions outside the CDRs, such as in one or more framework regions (FRs), such as FR1, FR2, FR3, and / or FR4.

[0575] In certain embodiments, the anti-MUC1 antibody of the present disclosure may comprise: a) a heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO:1; b) a light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO:2, 3, or 4.

[0576] In certain embodiments, an anti-MUC1 antibody of the present disclosure can comprise: a) a heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO:1 and a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs: 57-73; b) a light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO:2, 3, or 4.

[0577] In certain embodiments, an anti-MUC1 antibody of the present disclosure can comprise: a) a heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO:1 and a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs: 57-73, wherein the C present in the sequence LCTPSR in the constant region is replaced with fGly; b) a light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO:2, 3, or 4.

[0578] In certain embodiments, an anti-MUC1 antibody of the present disclosure can comprise: a) a heavy chain comprising a VH region having the amino acid sequence shown in SEQ ID NO:1 and a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs: 57-73, wherein the C present in the sequence LCTPSR in the constant region is replaced with fGly', where fGly' refers to an amino acid residue conjugated to a moiety of interest; b) a light chain comprising a VL region having the amino acid sequence shown in SEQ ID NO:2, 3, or 4.

[0579] In certain embodiments, an anti-MUC1 antibody of the present disclosure can comprise: a) a heavy chain comprising a VH region having an amino acid sequence that is 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 shown in SEQ ID NO:1, and a heavy chain constant region having an amino acid sequence that is 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 shown in any one of SEQ ID NOs: 57-73, wherein the C present in the sequence LCTPSR in the constant region is replaced with fGly', where fGly' refers to an amino acid residue conjugated to a moiety of interest; and b) a light chain comprising a VL region having an amino acid sequence that is 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 shown in SEQ ID NO:2, 3, or 4.

[0580] As measured by ELISA, the anti-MUC1 antibodies of the present disclosure can bind 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). The antibody concentration providing half-maximal response (e.g., half of the maximum fluorescence intensity) is measured as the EC50. MUC-1 can be the 20mer glycosylated MUC1 peptide as disclosed in Example 1.

[0581] As disclosed in Example 1, the anti-MUC1 antibodies of the present disclosure can bind the 20mer MUC1 glycosylated peptide but do not bind the recombinant 60mer MUC1 non-glycosylated peptide.

[0582] The anti-MUC1 antibodies of the present disclosure can bind cancerous tissues and can show no binding (e.g., insignificant binding as measured by immunohistochemistry or undetectable binding by immunohistochemistry) to normal tissues. For example, the anti-MUC1 antibodies described herein can bind human stomach, breast, and / or lung tissues with cancer cells while showing undetectable binding to human stomach, breast, and / or lung tissues without cancer cells.

[0583] The antibodies can be used in a variety of research, diagnostic, and therapeutic applications, including for performing any of the methods described in U.S. Patent Application Nos. US20120141375A1, US20160145343A1, the entire disclosures of which are incorporated herein by reference for all purposes.

[0584] A subject antibody specifically binds to a MUC1 polypeptide, wherein the epitope comprises amino acid residues within the human MUC1 antigen, the human MUC1 antigen comprising the amino acid sequence shown in SEQ ID NO:20:

[0585] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGS(SEQ ID NO:20)

[0586] In certain embodiments, the MUCl epitope to which the anti-MUCl antibodies disclosed herein bind is glycosylated.In certain embodiments, the MUCl epitope to which the anti-MUCl antibodies disclosed herein bind is present on MUCl expressed by epipulmonary adenocarcinoma cell lines and pulmonary epithelial cells.

[0587] The subject antibodies exhibit high affinity binding to MUC1. For example, the subject antibodies bind to MUC1 with a high affinity of at least about 10 -7 M, at least about 10 -8M, 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 greater than 10 -12 M binds to MUC1 with an affinity. The subject antibody binds to an epitope present on MUC1 with an affinity of about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, or about 10 -11 M to about 10 -12 M, or greater than 10 -12 M binds to an epitope present on MUC1.

[0588] In some cases, the anti-MUC1 antibodies of the present disclosure can induce apoptosis of cells that express MUC1 on their cell surface.

[0589] The "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.

[0590] As used herein, the term "immunoglobulin" refers to a protein composed of one or more polypeptides substantially encoded by immunoglobulin genes. Well-known human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region genes; and numerous immunoglobulin variable region genes. A full-length immunoglobulin light chain (about 25 kD or 214 amino acids) is encoded by a variable region gene at the N-terminus (about 110 amino acids) and a κ or λ constant region at the C-terminus. A full-length immunoglobulin heavy chain (about 50 kD or 446 amino acids) is encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other aforementioned constant region genes at the C-terminus (e.g., γ (encoding about 330 amino acids)). In some embodiments, the subject antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0591] In some embodiments, the subject antibody does not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but rather an antigen-binding fragment that comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain. In some embodiments, the antigen-binding fragment is comprised on separate polypeptide chains; in other embodiments, the antigen-binding fragment is comprised within a single polypeptide chain. As described above, the term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that are capable of specifically binding to MUC1. Examples of binding fragments include (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bond in the hinge region); (iii) Fd fragments (consisting of the VH and CH1 domains); (iv) Fv fragments (consisting of the VH and VL domains of a single arm of the antibody); (v) dAb fragments (consisting of the VH domain); (vi) isolated CDRs; (vii) single-chain Fv (scFv) (consisting of the VH and VL domains of a single arm of the antibody, the VH and VL domains being joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); (viii) diabodies (consisting of two scFvs, wherein the VH and VL domains are linked such that they do not pair to form a monovalent molecule; the VH of each scFv pair pairs with the VL domain of the other scFv to form a bivalent molecule); (ix) bispecific antibodies (consisting of at least two antigen-binding regions, each region binding a different epitope). In some embodiments, the subject antibody fragment is a Fab fragment. In some embodiments, the subject antibody fragment is a single-chain antibody (scFv).

[0592] In some embodiments, the subject antibody is a recombinant or modified antibody, such as a chimeric, humanized, deimmunized, or in vitro-generated antibody. As used herein, the term "recombinant" or "modified" antibody is intended to include all antibodies prepared, expressed, produced, 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 transgenic for human immunoglobulin genes (e.g., a mouse); (iv) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized, and in vitro-generated antibodies; and may optionally include constant regions derived from human germline immunoglobulin sequences.

[0593] Full-length bispecific antibodies can be generated, for example, by introducing substitutions at the heavy chain CH3 interface in each half-molecule to favor the formation of heterodimers between two antibody half-molecules with different specificities in a cell-free environment in vitro or using co-expression for Fab-arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies. 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 parental monospecific antibodies are reduced. The resulting free cysteine of one parental monospecific antibody forms an inter-heavy chain disulfide bond with the cysteine residue of the second parental monospecific antibody molecule, and simultaneously the CH3 domains of the parental antibodies are released and reformed by dissociation-association. The CH3 domains of the Fab arms can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each Fab arm or half-molecule binding a different epitope.

[0594] The "knob-in-hole" strategy (see, e.g., PCT International Publication No. WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the CH3 domain interface 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 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 a second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob". Exemplary CH3 substitution pairs that form the knob and hole (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S / L368A / Y407V.

[0595] Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by substituting a positively charged residue on one CH3 surface and a negatively charged residue on a second CH3 surface, as described in U.S. Patent Publication No. US2010 / 0015133; U.S. Patent Publication No. US2009 / 0182127; U.S. Patent Publication No. U82010 / 028637 or U.S. Patent Publication No. US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain): L351Y / F405A / Y407V / T394W, T366I / 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, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0596] 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 can be humanized. The constant regions, if present, can also be substantially or entirely from human immunoglobulins.

[0597] Methods for preparing humanized antibodies are known in the art. Replacing murine CDRs into the human variable domain framework can allow for retention of their correct spatial orientation, where for example the human variable domain framework adopts the same or a similar conformation as the murine variable framework (which is the source of the CDRs). This can be achieved by obtaining a human variable domain from a human antibody whose framework sequence exhibits a high degree of sequence identity with the murine variable framework domain (which is the source of the CDRs). The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a naturally occurring human antibody or can be a consensus sequence of several human antibodies.

[0598] After identifying the complementarity determining regions of the murine donor immunoglobulin and the appropriate human recipient immunoglobulin, the next step is to determine which residues, if any, in these components should be replaced to optimize the properties of the resulting humanized antibody. Generally, the replacement of human amino acid residues with murine amino acid residues should be minimized because the introduction of murine residues increases the risk of the antibody eliciting a human-anti-mouse-antibody (HAMA) response in humans. Methods for determining immune responses that are well recognized in the art can be implemented to monitor HAMA responses during a particular patient or clinical trial. Patients receiving the humanized antibody can be immunogenicity evaluated at the start of the therapy and throughout the administration. For example, using methods known in the art, including surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA assays, the HAMA response is measured by detecting antibodies against the humanized therapeutic reagent in serum samples from the patients. In many embodiments, the subject humanized antibody substantially does not elicit a HAMA response in human subjects.

[0599] Based on their possible effects on CDR conformation and / or binding to the antigen, certain amino acids from the human variable region framework residues are selected for replacement. The unnatural juxtaposition of the murine CDR regions with the human variable framework regions may result in unnatural conformational restraint, which, unless corrected by replacing certain amino acid residues, results in a loss of binding affinity. The selection of the amino acid residues for replacement 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, the molecular model is generated starting from the resolved 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 resolved three-dimensional structure, and the chain or domain showing greater sequence similarity is selected as the starting point for constructing 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 greater sequence identity are selected for modeling. The resolved starting structure is modified to allow for differences between the actual amino acids in the modeled immunoglobulin chain or domain 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 verifying that the distances between all atoms are within appropriate ranges and that the bond lengths and angles are within chemically acceptable limits.

[0600] When framework residues as defined by Kabat above constitute structural loop residues as defined by Chothia above, the amino acids present in the murine antibody can be selected for substitution into the humanized antibody. Residues of the "adjacent CDR region" include amino acid residues in positions adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., amino acid residues in positions adjacent to a CDR as defined by Kabat or a CDR as defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with the amino acids in the CDRs and, if selected from the acceptor, will distort the donor CDR and reduce affinity. In addition, adjacent amino acids can interact directly with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable for retaining all of the antigen contacts that provide affinity in the original antibody.

[0601] In some embodiments, the subject antibody comprises an scFv multimer. For example, in some embodiments, the subject antibody is an scFv dimer (e.g., comprising two tandem scFvs (scFv2)), an scFv trimer (e.g., comprising three tandem scFvs (scFv3)), an scFv tetramer (e.g., comprising four tandem scFvs (scFv4)), or a multimer of more than four scFvs (e.g., tandem). The scFv monomers can be tandemly linked by a linker having a length of from about 2 amino acids to about 10 amino acids (e.g., a length of 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids). Suitable linkers include, for example, (Gly) x (where x is an integer from 2 to 10), glycine-serine polymers, and the like.

[0602] In certain embodiments, the antibody is conjugated to a reagent via a cleavable or non-cleavable linker. Linkers suitable for use with the subject antibody include "flexible linkers". If present, the linker molecule generally has sufficient length to allow some flexible movement between the regions of attachment. The linker molecule is generally about 6-50 atoms in length. The linker molecule can also be, for example, aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Other linker molecules capable of binding to a polypeptide can be used according to the present disclosure.

[0603] According to some embodiments, the linker is a chemically labile linker, e.g., an acid-labile linker that is stable at neutral pH (blood pH 7.3 - 7.5) but hydrolyzes upon internalization into the mildly 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, etc. In certain embodiments, the linker is an enzyme-labile linker, e.g., an enzyme-labile linker that is stable in the bloodstream but is enzymatically cleaved upon internalization into target cells, e.g., by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells). Enzyme-labile linkers include, but are not limited to, linkers containing peptide bonds, e.g., dipeptide-based linkers, e.g., valine-citrulline (VC) linkers, e.g., maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linkers, valyl-alanyl-p-aminobenzyloxy (Val-Ala-PAB) linkers, etc.

[0604] In some embodiments, the 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 contain both light and heavy chain constant regions. 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 IgG1 Fc. The light chain constant region can be λ or κ. The subject antibody (e.g., the subject humanized antibody) can comprise sequences from more than one class or isotype. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as a single-chain antibody in which the heavy and light chain variable domains are linked by a spacer.

[0605] In some embodiments, the anti-MUC1 antibodies of the present disclosure can include one or more amino acid substitutions introduced into the Fc region. In some embodiments, the one or more amino acid substitutions can be at positions 239, 298, 326, 330, and 332 in the Fc region. In some embodiments, the anti-MUC1 antibodies of the present disclosure can 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.

[0606] In some embodiments, the subject antibody comprises a free thiol (-SH) group at the carboxyl terminus, wherein the free thiol group can be used to link the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, etc.

[0607] In some embodiments, the subject antibody comprises one or more non-naturally occurring amino acids. In some embodiments, the non-naturally encoded amino acids comprise a carbonyl group, an acetyl group, an aminooxy group, a hydrazino 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 linkage to a polymer, a second polypeptide, a scaffold, etc. 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.

[0608] The present disclosure also provides anti-MUC1 antibodies having a linked moiety of interest (e.g., a detectable label, a drug, a half-life extending moiety, etc.). Modification of the antibody can be accomplished by a variety of synthetic and / or recombinant methods. Linking one or more moieties to the antibody can provide one or more of a 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, including 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); water-soluble polymers (e.g., polyethylene glycolylation); purification tags (e.g., facilitating separation by affinity chromatography (e.g., linkage of a FLAG epitope); membrane localization domains (e.g., lipid or glycosylphosphatidylinositol (GPI)-type anchors); immobilization tags (e.g., to facilitate polypeptide attachment to a surface, including selective attachment); drugs (e.g., facilitating drug targeting by linking a drug to the antibody), etc.

[0609] In some embodiments, the subject antibody is linked (e.g., covalently) 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 straight-chain 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 referred to 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; polyorthoester; polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphate ester; polyphosphate ester urethane; poly(amino acid); cyanoacrylate; poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-ester) (e.g., poly(ethylene oxide)-poly(lactic acid) (PEO / PLA) copolymer); polyalkylene oxalate; polyphosphazene; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethane; silicone; polyester; polyolefin; polyisobutylene and ethylene-α-olefin copolymer; acrylic polymer and copolymer; vinyl halide polymer and copolymer, such as polyvinyl chloride; polyvinyl ether, such as polyvinyl methyl ether; polyvinylidene halide, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyvinyl aromatic compound, such as polystyrene; polyvinyl ester, such as polyvinyl acetate; copolymer of vinyl monomers and copolymer of vinyl monomers and olefins, such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, ethylene-vinyl acetate copolymer; polyamide, such as nylon 66 and polycaprolactam; alkyd resin; polycarbonate; polyformaldehyde; polyimide; polyether; epoxy resin; polyurethane; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ether; amorphous Teflon; poly(ethylene glycol), and carboxymethyl cellulose.

[0610] Suitable synthetic polymers include unsubstituted and substituted straight-chain or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, such as substituted poly(ethylene glycol), such as methoxy poly(ethylene glycol), and their derivatives. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen, and their derivatives.

[0611] The average molecular weight of the suitable polymer can range from 500 Da to 50,000 Da, such as from 5,000 Da to 40,000 Da, or from 25,000 to 40,000 Da. For example, in some embodiments, when the subject antibody comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the molecular weight of the PEG or methoxypoly(ethylene glycol) polymer can be in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0612] In some embodiments, the subject antibody is covalently linked to a PEG polymer. In some embodiments, the subject scFv multimer is covalently linked to a PEG polymer. PEGs suitable for conjugation to proteins are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2) n O-R, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and where n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. The PEG conjugated to the subject antibody can be branched or branched. Branched PEG derivatives include derivatives of star PEGs and multi-arm PEGs.

[0613] The subject antibody can be glycosylated, for example, the subject antibody can comprise a covalently linked carbohydrate or polysaccharide moiety. Glycosylation of antibodies is generally N-linked or O-linked. Glycosylation sites can be conveniently added to the antibody by altering the amino acid sequence to contain N- or O-linked glycosylation sites. Similarly, removal of glycosylation sites can be achieved by amino acid alterations within the native glycosylation sites of the antibody.

[0614] The subject antibody can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the subject antibody, a synthetic polymer, a carbohydrate, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides through the amino moiety of the polypeptide. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional thiol-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 of a mixture of polypeptides to be linked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At a mild alkaline pH, imidoesters react only with primary amines to form imidoamides and do not affect the total charge of the crosslinked polypeptide. Homobifunctional thiol-reactive crosslinkers include bis(maleimido)hexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-bis-(3',2'-pyridyldithio)propionamidobutane (DPDPB).

[0615] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a thiol-reactive moiety) and crosslink to one of the polypeptides through the amine or thiol-reactive moiety and then react with the other polypeptide through the unreacted moiety. A variety of heterobifunctional haloacetyl crosslinkers are available, and pyridyldisulfide crosslinkers are also available. Carbodiimides are classic examples of heterobifunctional crosslinkers used to couple a carboxyl group to an amine to form an amide bond.

[0616] In some embodiments, the subject antibody will 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 of skill in the art. The most common radiopaque materials include iodides, bromides, or barium salts. Other radiopaque materials are also known and include, but are not limited to, organic bismuth derivatives, radiopaque polyurethanes, organic bismuth complexes, radiopaque barium polycomplexes, etc.

[0617] In some embodiments, the subject antibody comprises a polyamine modification. The subject antibody can be modified with a naturally occurring or synthetic polyamine. Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1,3-diaminopropane, norspermidine, syn-homospermidine, thermospermine, thermospermidine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines have the empirical formula C X H Y NZ It consists of a hydrocarbon chain having 3 to 12 carbon atoms, which can be cyclic or acyclic, branched or unbranched, and further includes 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 crosslinking method.

[0618] When the anti-MUC1 antibody of the present disclosure contains a covalently linked heterologous moiety, the heterologous moiety can be linked directly or through a linker to the anti-MUC1 heavy chain and / or light chain. Suitable linkers can be readily selected and can have any suitable different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0619] Examples of flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS) n , (GSGGS) n (SEQ ID NO:21) and (GGGS) n (SEQ ID NO:22), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art.

[0620] Antibody modification methods

[0621] The anti-MUC1 antibody conjugates of the present disclosure can include: 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 include VH and / or VL domains conjugated to a moiety of interest.

[0622] In one instance, an antibody can be modified to include 2-formylglycine residues, which can serve as chemical handles for attaching heterologous moieties. For example, the heavy and / or light chain constant regions of the anti-MUC1 of the present disclosure can be modified to include an amino acid sequence containing 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. FGE acts in a sequence-specific manner, i.e., FGE acts on the sulfatase motif located within the immunoglobulin polypeptide. The moiety of interest is provided as a component of the reaction participant for an aldehyde reaction with the fGly residue of the converted aldehyde tag of the labeled Ig polypeptide. A variety of commercially available reagents can be used to effect the attachment of the moiety of interest to the fGly residue of the aldehyde-labeled Ig polypeptide. For example, the alkoxyamine, hydrazide, or thiosemicarbazide derivatives of many moieties of interest are suitable reaction participants and are either available or can be generated using standard chemical methods.

[0623] As described above, the amino acid sequence of the anti-MUC1 antibody can be modified to include a sulfatase motif containing a serine or cysteine residue that can be converted (oxidized) to a 2-formylglycine (fGly) residue by the action of 2-formylglycine-generating enzyme (FGE) in vivo (e.g., when translating a protein containing an aldehyde tag in a cell) or in vitro (e.g., by contacting a protein containing an aldehyde tag with FGE in a cell-free system). Such a sulfatase motif may also be referred to herein as an FGE modification site.

[0624] Sulfatase motif

[0625] The length of the minimal sulfatase motif of the aldehyde tag is typically 5 or 6 amino acid residues and generally does not exceed 6 amino acid residues. The sulfatase motif provided in the Ig polypeptide is at least 5 or 6 amino acid residues and can be, for example, 5 to 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 to define a sulfatase motif having a length less than 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acid residues.

[0626] In certain embodiments, the polypeptide of interest comprises an amino acid sequence in which one or more amino acid residues, e.g., 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide comprises a modification (insertion, addition, deletion, and / or substitution / replacement) of the amino acid sequence that is less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues relative to the native amino acid sequence of the polypeptide. When the native amino acid sequence of the polypeptide (e.g., an anti-MUC1 antibody) contains one or more residues of the desired sulfatase motif, the total number of modified residues can be reduced, e.g., by site-specific modification (insertion, addition, deletion, substitution / replacement) of amino acid residues flanking the native amino acid residue to provide the sequence of the desired sulfatase motif. In certain embodiments, the degree of modification of the native amino acid sequence of the target anti-MUC1 polypeptide is minimized such that the number of amino acid residues inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus) is minimized. Minimizing the degree of modification of the amino acid sequence of the target anti-MUC1 polypeptide can minimize the effect that such modifications may have on anti-MUC1 function and / or structure.

[0627] It should be noted that while aldehyde tags of particular interest are those that contain at least one minimal sulfatase motif (also referred to as a “consensus sulfatase motif”), it will be readily understood that longer aldehyde tags are contemplated and encompassed by the present disclosure and can be used in the compositions and methods of the present disclosure. Thus, an aldehyde tag can contain a minimal sulfatase motif having 5 or 6 residues, or can be longer and contain a minimal sulfatase motif with additional amino acid residues flanking the N- and / or C-terminus. Aldehyde tags of, e.g., 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues.

[0628] An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; for example, the aldehyde tag can be present 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 present within the CH1 domain of the Ig heavy chain. The aldehyde tag can be present within the CH2 domain of the Ig heavy chain. The aldehyde tag can be present within the CH3 domain of the Ig heavy chain. The aldehyde tag can be present within the constant region of an Ig light chain, for example, within the κ light chain constant region or the λ light chain constant region.

[0629] In certain embodiments, the sulfatase motif used can be described by the following formula:

[0630] X 1 Z 1 X 2 Z 2 X 3 Z 3 (I'), where

[0631] Z 1 is cysteine or serine (which can also be represented as (C / S));

[0632] Z 2 is a proline or alanine residue (which can also be represented as (P / A));

[0633] Z 3 is a basic amino acid (e.g., arginine (R), and can 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;

[0634] X 1 is present or absent, and when present, can be any amino acid, but is usually an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than aromatic or charged amino acids), usually L, M, V, S, or T, more usually L, M, S, or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 is present; and

[0635] X 2 and X 3Independently can be any amino acid, but is typically an aliphatic amino acid, a polar uncharged amino acid, or a sulfur-containing amino acid (e.g., excluding aromatic or charged amino acids), such as, for example, S, T, A, V, G, or C; for example, S, T, A, V, or G. In one instance, the aldehyde tag has the formula L(C / S)TPSR (SEQ ID NO:5), such as LCTPSR (SEQ ID NO:6) or LSTPSR (SEQ ID NO:23). Accordingly, the present disclosure provides antibodies comprising an aldehyde-labeled Ig heavy chain and / or an aldehyde-labeled Ig light chain, wherein the aldehyde-labeled Ig antibody comprises an Ig constant region amino acid sequence of the heavy chain and / or light chain containing such a sulfatase motif.

[0636] For example, in some embodiments, the amino acid sequence of the anti-MUC1 heavy chain and / or light chain can be modified to provide a sequence of at least 5 amino acids having the formula X 1 Z 1 X 2 Z 2 X 3 Z 3 wherein

[0637] Z 1 is cysteine or serine;

[0638] Z 2 is a proline or alanine residue;

[0639] Z 3 is an aliphatic or basic amino acid;

[0640] X 1 is present or absent, and when present, is any amino acid, provided that when the heterologous sulfatase motif is located at the N-terminus of the polypeptide, X 1 is present;

[0641] X 2 and X 3 are each independently any amino acid,

[0642] wherein the sequence is within or near a solvent-accessible loop region of the Ig constant region and wherein the sequence is not at the C-terminus of the Ig heavy chain.

[0643] The sulfatase motif is typically selected to be convertible by the selected FGE (e.g., the FGE present in the host cell expressing the aldehyde-labeled polypeptide or the FGE contacted with the aldehyde-labeled polypeptide in a cell-free in vitro method).

[0644] For example, when the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including human FGE), the sulfatase motif can have the following formula:

[0645] X 1 CX 2 PX 3 Z 3 (I”)

[0646] wherein

[0647] X 1 may or may not be present, and when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (e.g., other than aromatic or charged amino acids), such as L, M, S or V, provided that when the sulfatase motif is located at the N-terminus of the target polypeptide, X 1 is present;

[0648] X 2 and X 3 independently may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (e.g., other than aromatic or charged amino acids), such as, S, T, A, V, G or C, such as, S, T, A, V or G; and

[0649] Z 3 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), such as lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), such as A, G, L, V or I.

[0650] 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).

[0651] Sequence Containing fGly

[0652] Generally, the FGE for promoting the conversion of cysteine or serine in the su...

Claims

1. A conjugate of formula (I): wherein Z is CR 4 or N; R 1 selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group; R 2 and R 3 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, or R 2 and R 3 optionally cyclically linked to form a 5- or 6-membered heterocyclic group; 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group; L is a linker; W 1 is a drug; and W 2 is an anti-MUC1 antibody.

2. The conjugate according to claim 1, wherein L comprises: -(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 -, wherein a, b, c, d, e, and f are each independently 0 or 1, where 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 are each independently selected from a covalent bond, (C1-C 12 )alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (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-benzylamine (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, where EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analogue, where 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 are each independently selected from 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 SO2- 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 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, heterocyclic group and substituted heterocyclic group.

3. The conjugate according to claim 2, wherein: T 1 selected from (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 2 、T 3 、T 4 、T 5 and T 6 are each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal group, hydrazine and ester; and V 1 、V 2 、V 3 、V 4 、V 5 and V 6 are each independently selected from 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 SO2- and -P(O)OH-; wherein: (PEG) n where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: wherein y is an integer from 1 to 6, and r is 0 or 1; 4-Amino-piperidine (4AP) is and Each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, where any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.

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

6. The conjugate according to any one of claims 2 to 5, wherein: T 1 is (C1-C 12 ) alkyl and V 1 is -CO-; T 2 is an amino acid analogue and V 2 is -NH-; T 3 is (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; and f is 0.

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

8. The conjugate according to any one of claims 1 to 7, wherein the conjugate has the following structure:

9. A conjugate of formula (II): wherein: Z 1 , Z 2 , Z 3 and Z 4 Each independently selected from CR 24 , N and CL B -W 12 , where at least one Z 1 , Z 2 , Z 3 and Z 4 For CL B -W 12 ; R 21 selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group; R 22 and R 23 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, or R 22 and R 23 optionally joined cyclically to form a 5 - or 6 - membered heterocyclic group; 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, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group; L A is the first joint; L B is the second joint; W 11 is the first drug; W 12 is the second drug; and W 13 is an anti-MUC1 antibody.

10. The conjugate according to claim 9, wherein Z 1 is CR 24 .

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

12. The conjugate according to claim 9, wherein Z 3 is C-L B -W 12 .

13. The conjugate according to any one of claims 9 to 12, wherein L A comprises: -(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 -, wherein 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 are each independently selected from a covalent bond, (C1-C 12 )alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (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-benzylamine (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide and ester, where EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analogue, where 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 are each independently selected from 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 SO2- 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; 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, heterocyclic group, and substituted heterocyclic group.

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

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

16. The conjugate according to any one of claims 9 to 15, wherein: T 1 is (C1-C 12 ) alkyl and V 1 is -CONH-; T 2 is a substituted (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 both e and f are 0.

17. The conjugate according to any one of claims 9 to 16, wherein L B comprises: -(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; T 7 、T 8 、T 9 、T 10 、T 11 、T 12 and T 13 are each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic group and substituted heterocyclic group, (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-benzylamine (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide and ester, where EDA is an ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analogue, where 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 are each independently selected from 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 SO2- 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; 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, heterocyclic group, and substituted heterocyclic group.

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

19. The conjugate according to any one of claims 17 to 18, wherein the glycoside is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

20. The conjugate according to any one of claims 17 to 19, wherein: T 7 does not exist and V 7 is -NHCO-; T 8 is a (C1-C 12 ) alkyl and V 8 is -CONH-; T 9 is a substituted (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; and both l and m are 0.

21. The conjugate according to claim 9, wherein the conjugate has the following structure:

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

23. The conjugate according to claim 22, wherein the anti-MUC1 antibody is an IgG1κ antibody.

24. The conjugate according to any one of claims 1 to 23, wherein the anti-MUC1 antibody comprises a sequence of formula (III): X 1 (fGly’)X 2 Z 2 X 3 Z 3 (III), wherein X 1 is present or absent, and when present, can be any amino acid, provided that when the sequence is at the N-terminus of the conjugate, X 1 is present; fGly' is an amino acid residue conjugated to the drug via a linker; X 2 and X 3 each independently is any amino acid; Z 2 is a proline or alanine residue; and Z 3 is an alkaline amino acid or an aliphatic amino acid.

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

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

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

28. The conjugate according to claim 27, wherein the heavy chain constant region comprises the sequence of formula (III): X 1 (fGly’)X 2 Z 2 X 3 Z 3 (III), wherein X 1 is present or absent, and when present, can be any amino acid, provided that when the sequence is at the N-terminus of the conjugate, X 1 is present; fGly' is an amino acid residue conjugated to the drug through a linker; X 2 and X 3 each independently is any amino acid; Z 2 is a proline or alanine residue; Z 3 is an alkaline amino acid or an aliphatic amino acid, and wherein the sequence is the C-terminus of the amino acid sequence SLSLSPG (SEQ ID NO:187).

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

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

31. The conjugate according to 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 shown in SEQ ID NO:57, and comprises an fGly' residue at amino acid position 332 instead of C.

32. The conjugate according to 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 any one of the amino acid sequences shown in SEQ ID NO:58, 62, 66 and 70, and comprises an fGly' residue at amino acid position 59 instead of C.

33. The conjugate according to 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 any one of the amino acid sequences shown in SEQ ID NO:59, 63, 67 and 71, and comprises an fGly' residue at amino acid position 62 instead of C.

34. The conjugate according to 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 any one of the amino acid sequences shown in SEQ ID NO:60, 64, 68 and 72, and comprises an fGly’ residue at amino acid position 92 instead of C.

35. The conjugate according to 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 shown 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 according to 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 conjugate according to claim 36, wherein the light chain constant region comprises the sequence of formula (III): X 1 (fGly’)X 2 Z 2 X 3 Z 3 (III) wherein X 1 is present or absent, and when present, can be any amino acid, provided that when the sequence is at the N-terminus of the conjugate, X 1 is present; fGly' is an amino acid residue conjugated to the drug through a linker; X 2 and X 3 each independently is any amino acid; Z 2 is a proline or alanine residue; Z 3 is an alkaline amino acid or an aliphatic amino acid, and wherein the sequence is the C-terminus of the amino acid sequence KVDNAL (SEQ ID NO: 101) and / or the N-terminus of the sequence QSGNSQ (SEQ ID NO: 102).

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

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

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

41. The conjugate according to claim 40, wherein the light chain constant region comprises the sequence of formula (III): X 1 (fGly’)X 2 Z 2 X 3 Z 3 (III) wherein X 1 is present or absent, and when present, can be any amino acid, provided that when the sequence is at the N-terminus of the conjugate, X 1 is present; fGly' is an amino acid residue conjugated to the drug through a linker; X 2 and X 3 each independently is any amino acid; Z 2 is a proline or alanine residue; Z 3 is an alkaline amino acid or an aliphatic amino acid, and wherein the sequence is the C-terminus of the amino acid sequence SWNSGA (SEQ ID NO: 104) and / or the N-terminus of the amino acid sequence GVHTFP (SEQ ID NO: 105).

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

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

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

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

46. The conjugate according to any one of claims 1 to 45, wherein the anti-MUC1 antibody competes with an anti-MUC1 antibody comprising: a variable heavy (VH) chain that comprises heavy chain complementarity determining regions 1-3 (HCDR 1-3) of a VH chain having the following sequence: EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRP GKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSS LRSEDTAVYYCARGLRYALDYWGQGTLVTVSS(SEQ ID NO:1); and a variable light (VL) chain comprising light chain complementarity determining regions 1-3 (LCDR 1-3) of a VL chain having the following sequence: EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQA PRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCH QYAWSPPTFGQGTKLEIK(SEQ ID NO:2); EIVLTQSPATLSLSPGERATLSCRASSSVGSSNLYWYQQKPGQ APRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYC HQYRWSPPTFGQGTKLEIK(SEQ ID NO:3); or EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQA PRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQ YSWSPPTFGQGTKLEIK(SEQ ID NO:4).

47. The conjugate according to any one of claims 1 to 45, wherein the anti-MUC1 antibody comprises: a variable heavy (VH) chain comprising heavy chain complementarity determining regions 1-3 (HCDR 1-3) of a VH chain having the following sequence: EVQLVQSGAEVKKPGATVKISCKVSGYTFTDHTMHWIKQRP GKGLEWMGYFYPRDDSTNYNEKFKGRVTLTADKSTDTAYMELSS LRSEDTAVYYCARGLRYALDYWGQGTLVTVSS(SEQ ID NO:1); and a variable light (VL) chain comprising light chain complementarity determining regions 1-3 (LCDR 1-3) of a VL chain having the following sequence: EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQA PRLWIYGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCH QYAWSPPTFGQGTKLEIK(SEQ ID NO:2); EIVLTQSPATLSLSPGERATLSCRASSSVGSSNLYWYQQKPGQAPRLWIYRSTKLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYRWSPPTFGQGTKLEIK (SEQ ID NO:3); or EIVLTQSPATLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQAPRLWIIGTSNLASGVPARFSGSGSGTDYTLTISSLEPEDAAVYYCHQYSWSPPTFGQGTKLEIK (SEQ ID NO:4).

48. The conjugate according to claim 46 or 47, wherein the VH polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown 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. The conjugate according to any one of claims 46 to 49, wherein: HCDR1 comprises the amino acid sequence DHTMH (SEQ ID NO:17); HCDR2 comprises the amino acid sequence YFYPRDDSTNYNEKFKG (SEQ ID NO:18); HCDR3 comprises the amino acid sequence GLRYALDY (SEQ ID NO:9); LCDR1 comprises the amino acid sequence RASSSVSSSYLY (SEQ ID NO:10); LCDR2 comprises the amino acid sequence GTSNLAS (SEQ ID NO:11); and LCDR3 comprises the amino acid sequence HQYAWSPPT (SEQ ID NO:12), according to Kabat definition.

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

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

53. A pharmaceutical composition comprising: a conjugate according to any one of claims 1 to 52; and a pharmaceutically acceptable excipient.

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

55. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 53, wherein the administration effectively treats the cancer in the subject.

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

57. The method according to claim 56, wherein the cancer is characterized by cancer cells expressing glycosylated MUC1.

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

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

60. The method according to claim 59, wherein the triple negative breast cancer is metastatic triple negative breast cancer.

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

62. A method of delivering a drug to a target in a subject, the method comprising: administering to the subject the pharmaceutical composition according to claim 53, wherein the administration effectively releases a therapeutically effective amount of the drug from the conjugate at the target in the subject.

Images