Antibodies and antibody conjugates specific for nectin-4 and methods of use thereof

Abstract

The present disclosure provides antibodies specific to Nectin-4, and antibody conjugates, such as antibody-drug conjugates (ADCs), comprising such antibodies. The present disclosure also encompasses methods for producing such antibodies and antibody conjugates, as well as methods for using such antibodies and antibody conjugates. Also provided are compositions comprising the antibodies and antibody conjugates of the present disclosure, including, in some cases, pharmaceutical compositions. In one embodiment, a method of using the ADC is provided, comprising administering a therapeutically effective amount of an antibody or antibody conjugate of the present disclosure to an individual with a cell proliferative disorder.

Description

[Technical Field]

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

[0002] The field of protein-small molecule conjugates has seen remarkable progress, resulting in a large number of clinically beneficial drugs, with many more expected to be developed in the future. Protein conjugate therapeutics may offer several advantages, including specificity, functional diversity, and relatively low off-target activity, resulting in fewer side effects. Chemical modification of proteins may enhance these advantages by making protein conjugate therapeutics more potent, more stable, or more versatile.

[0003] Many standard chemical transformations are commonly used to create and manipulate post-translational modifications on proteins. Many methods are available to selectively modify the side chains of certain amino acids. For example, carboxylic acid side chains (aspartic acid and glutamic acid) may be targeted by first activating them with water-soluble carbodiimide reagents and then reacting them with amines. Similarly, lysines can be targeted through the use of activated esters or isothiocyanates, and cysteine ​​thiols can be targeted with maleimides and α-halo-carbonyls.

[0004] One major obstacle to creating chemically altered protein therapeutics or reagents is producing the protein in a biologically active, homogenous form. Conjugating a drug or detectable label to a polypeptide can be difficult to control, resulting in a heterogeneous mixture of complexes that vary in the number of drug molecules attached and the location of the chemical bond. In some cases, it may be desirable to control the location of attachment and / or the drug or detectable label attached to the polypeptide using synthetic organic chemistry tools that direct precise and selective chemical bond formation on the polypeptide.

[0005] Nectin-4 (also known as nectin cell adhesion molecule 4) is a member of the nectin family. Nectin-4 is a type I transmembrane protein and a member of the nectin adhesion protein family. Nectin adhesion proteins are structurally related and exhibit three conserved immunoglobulin-like domains (V, C, and C) in their extracellular regions. Nectin-4 has a molecular weight of approximately 55 kDa, with the molecular weight of the extracellular domain being approximately 36 kDa.

[0006] Nectin-4 can form homodimers or heterodimers with nectin-1. Nectin-4 regulates several cellular activities, including migration, proliferation, differentiation, polarization, and viral entry. While other nectin family members are widely expressed in adult tissues, nectin-4 is primarily restricted to the embryo and placenta. Furthermore, nectin-4 is overexpressed in various solid tumors, including ovarian cancer, breast ductal carcinoma, lung adenocarcinoma, and pancreatic cancer. Nectin-4 has emerged as a metastasis-associated protein and may be associated with disease progression and poor prognosis. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides antibodies specific to Nectin-4 and antibody conjugates (e.g., antibody-drug conjugates (ADCs)) comprising such antibodies. The present disclosure also encompasses methods for producing such antibodies and antibody conjugates, as well as methods for using such antibodies and antibody conjugates. Each embodiment is described in more detail in the following sections. Compositions comprising the antibodies and ADCs of the present disclosure are also provided, and in some cases, include pharmaceutical compositions. In one aspect, a method of using an ADC of the present disclosure is provided, comprising administering a therapeutically effective amount of the ADC to an individual with a cell proliferative disorder.

[0008] A brief description of the sequence SEQ ID NOs: 1-17: Heavy chains of the antibodies disclosed herein.

[0009] SEQ ID NOs: 18-31: Light chains of antibodies disclosed herein.

[0010] SEQ ID NOs: 32-69: Heavy and light chain CDRs of the antibodies disclosed herein.

[0011] SEQ ID NOs: 70-86: Heavy chain constant regions of the antibodies disclosed herein.

[0012] SEQ ID NOs: 87 and 88: IgG1 heavy and light chains.

[0013] SEQ ID NOs: 89-93: Heavy chain constant regions of different Ig isotypes.

[0014] SEQ ID NOs: 94-98: Light chain constant regions of different types and organisms.

[0015] SEQ ID NO: 99: Sequence of human nectin-4 protein.

[0016] SEQ ID NOs: 100-101: Flexible linkers comprising glycine polymers.

[0017] SEQ ID NOs: 102-126: Examples of sulfatase motifs before conversion by formylglycine generating enzyme (FGE).

[0018] SEQ ID NOs: 127-128 and 245-246: Examples of sulfatase motifs after conversion by FGE.

[0019] SEQ ID NO: 129: Amino acid sequence within the heavy chain constant region into which the sulfatase motif has been inserted.

[0020] SEQ ID NOs: 130 to 244: Sequences within the constant regions of various immunoglobulins. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the binding of chimeric anti-nectin-4 antibodies to recombinant human nectin-4 protein. [Figure 2] FIG. 2 shows the binding of chimeric anti-nectin-4 antibodies to recombinant human nectin-4 protein. [Figure 3] FIG. 3 shows the binding of anti-nectin-4 antibodies to human nectin family members and human Necl family members. [Figure 4] FIG. 4 shows the binding of variants of the 12E11 antibody clone to human nectin family members and human Necl family members. [Figure 5] FIG. 5 shows the binding of variants of the 12E11 antibody clone to human nectin family members and human NecI family members. [Figure 6] FIG. 6 shows the binding of humanized 5D9 variants to human nectin-4. [Figure 7] FIG. 7 shows the binding of humanized 5D9 variants to human nectin-4. [Figure 8] FIG. 8 shows the binding of humanized 5D9 variants to human nectin-4. [Figure 9] FIG. 9 shows the binding of humanized 5D9 variants to human nectin-4. [Figure 10]FIG. 10 shows the in vitro efficacy of chimeric anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 11] FIG. 11 shows the in vitro efficacy of chimeric anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 12] Figure 12 shows the in vitro efficacy of chimeric anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 13] Figure 13 shows the in vitro efficacy of chimeric anti-nectin-4 ADCs or controls on HEK cells overexpressing human nectin-4. [Figure 14] Figure 14 shows the in vitro efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 15] FIG. 15 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls on SK-BR-3 cells in vitro. [Figure 16] FIG. 16 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADC or control on MDA-MB-468 cells in vitro. [Figure 17] Figure 17 shows the in vitro efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 18] FIG. 18 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADC or control on SK-BR-3 cells in vitro. [Figure 19] Figure 19. In vitro efficacy of humanized 5D9 variant anti-nectin-4 ADC or control on MDA-MB-468 cells. [Figure 20]Figure 20 shows the in vitro efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 21] FIG. 21 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls on SK-BR-3 cells in vitro. [Figure 22] FIG. 22 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls on MDA-MB-468 cells in vitro. [Figure 23] Figure 23 shows the in vitro efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 24] FIG. 24 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls on SK-BR-3 cells in vitro. [Figure 25] FIG. 25 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADC or control on MDA-MB-468 cells in vitro. [Figure 26] Figure 26 shows the in vitro efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls against HEK cells overexpressing human nectin-4. [Figure 27] FIG. 27 shows the efficacy of h5D9 variant anti-nectin-4 ADC or control on SK-BR-3 cells in vitro. [Figure 28] FIG. 28 shows the efficacy of humanized 5D9 variant anti-nectin-4 ADCs or controls on MDA-MB-468 cells in vitro. [Figure 29] FIG. 29 shows the in vivo efficacy of dual-tagged, aldehyde-tagged enfortumab antibody conjugated to Compound 20 on the NCI-H1781 xenograft model. [Figure 30A] Figure 30A shows a site map indicating potential modification sites for generating aldehyde-tagged Ig polypeptides. The upper sequence is the amino acid sequence of a conserved region of an IgG1 light chain polypeptide (SEQ ID NO: 87), indicating potential modification sites within the Ig light chain; the lower sequence is the amino acid sequence of a conserved region of an Ig heavy chain polypeptide (SEQ ID NO: 88) (GenBank accession number AAG00909), indicating potential modification sites within the Ig heavy chain. The numbering of the heavy and light chains is based on the full-length heavy and light chains. [Figure 30B] Figure 30B shows an alignment of the human immunoglobulin heavy chain constant region of IgG1 (SEQ ID NO: 89; GenBank P01857.1), the human immunoglobulin heavy chain constant region of IgG2 (SEQ ID NO: 90; GenBank P01859.2), the human immunoglobulin heavy chain constant region of IgG3 (SEQ ID NO: 91; GenBank P01860.2), the human immunoglobulin heavy chain constant region of IgG4 (SEQ ID NO: 92; GenBank AAB59394.1), and the human immunoglobulin heavy chain constant region of IgA (SEQ ID NO: 93; GenBank AAAT74070), showing modification sites within the immunoglobulin heavy chain that can accommodate aldehyde tags. The numbering of the heavy and light chains is based on the complete heavy and light chains. [Figure 30C] Figure 30C shows an alignment of immunoglobulin light chain constant regions, showing possible modification sites within the immunoglobulin light chain to provide an aldehyde tag. Sequence 1 = human kappa light chain constant region; GenBank CAA75031.1; SEQ ID NO: 94. Sequence 2 = human kappa light chain constant region; GenBank BAC0168.1; SEQ ID NO: 95. Sequence 3 = human lambda light chain constant region; GenBank CAA75033; SEQ ID NO: 96. Sequence 4 = Mus musculus light chain constant region; GenBank AAB09710.1; SEQ ID NO: 97. Sequence 5 = Rattus norvegicus light chain constant region; GenBank AAD10133; SEQ ID NO: 98. [Figure 30D]Figure 30D shows an alignment of immunoglobulin light chain constant regions, showing possible modification sites within the immunoglobulin light chain to provide an aldehyde tag. Sequence 1 = human kappa light chain constant region; GenBank CAA75031.1; SEQ ID NO: 52. Sequence 2 = human kappa light chain constant region; GenBank BAC0168.1; SEQ ID NO: 53. Sequence 3 = human lambda light chain constant region; GenBank CAA75033; SEQ ID NO: 54. Sequence 4 = Mus musculus light chain constant region; GenBank AAB09710.1; SEQ ID NO: 55. Sequence 5 = Rattus norvegicus light chain constant region; GenBank AAD10133; SEQ ID NO: 56. [Figure 31] Figure 31 shows a graph from the NCI-H1781 xenograft study in which the listed anti-Nectin-4 ADCs were administered intravenously in a single dose of 2.5 mg / kg or 7.5 mg / kg on day 0. VH4 / VL1 Compound 8 (RED-601) and VH4 / VL5 Compound 8 both utilize an internal 91N tag and deliver half the payload dose compared to padoseb. The isotype control ADC had minimal activity. [Figure 32] Figure 32 shows a graph of the NCI-H1781 xenograft study in which the listed anti-Nectin-4 ADC or isotype ADC was administered intravenously at a single dose of 2.5 mg / kg or 7.5 mg / kg on day 0. VH4 / VL1 compound 25 (RED-694) was engineered with a 91N tag in a DAR4 format and with a 91N / 116E dual tag combination in a DAR8 format. Padoseb (generic) was included as a comparison. The isotype control compound 25 ADC had minimal activity. [Figure 33]Figure 33 shows a graph of the NCI-H1781 xenograft study in which the listed anti-Nectin-4 ADC or isotype ADC was administered intravenously at a single dose of 2.5 mg / kg or 7.5 mg / kg on day 0. VH4 / VL5 compound 25 (RED-694) was engineered with a DAR of 4 using the 91N tag and a DAR of 8 using the 91N / 116E dual tag combination. Padoseb (generic) was included as a comparison. The isotype control compound 25 ADC had minimal activity. [Figure 34] Figure 34. Dual-tagged Nectin-4 VH4 / VL1 antibody conjugated to compound 8 results in a DAR of 3.74 as measured by PLRP. [Figure 35] Figure 35. Dual-tagged Nectin-4 VH4 / VL1 antibody conjugated to Compound 8 is 98.5% monomeric as determined by SEC. [Figure 36] Figure 36. Dual-tagged Nectin-4 VH4 / VL5 antibody conjugated to Compound 8 results in a DAR of 3.73 as measured by PLRP. [Figure 37] Figure 37. Dual-tagged Nectin-4 VH4 / VL5 antibody conjugated to Compound 8 is 98.0% monomeric as determined by SEC. [Figure 38] Figure 38. Dual-tagged Nectin-4 VH4 / VL1 antibody conjugated to compound 25 results in a DAR of 6.89 as measured by PLRP. [Figure 39] Figure 39. Dual-tagged Nectin-4 VH4 / VL1 antibody conjugated to Compound 25 is 98.7% monomeric as determined by SEC. [Figure 40] Figure 40. Dual-tagged Nectin-4 VH4 / VL5 antibody conjugated to compound 25 results in a DAR of 6.86 as measured by PLRP. [Figure 41] Figure 41. Dual-tagged Nectin-4 VH4 / VL5 antibody conjugated to Compound 25 is 96.6% monomeric as determined by SEC. [Figure 42] Figure 42. Single-tagged Nectin-4 VH4 / VL1 antibody conjugated to compound 25 results in a DAR of 3.16 as measured by PLRP. [Figure 43] Figure 43. Single-tagged Nectin-4 VH4 / VL1 antibody conjugated to Compound 25 is 97.2% monomeric as determined by SEC. [Figure 44] Figure 44. Single-tagged Nectin-4 VH4 / VL5 antibody conjugated to Compound 25 results in a DAR of 3.25 as measured by PLRP. [Figure 45] Figure 45. Single-tagged Nectin-4 VH4 / VL5 antibody conjugated to Compound 25 results in a DAR of 3.25 as measured by PLRP. [Figure 46] Figure 46. Clinical findings in rats repeatedly administered a rat cross-reactive Nectin-4 ADC. Arrows indicate the days of administration. No findings were observed in animals administered the Compound 25 conjugate, while the clinical findings in the Vedotin-treated group averaged 2.5 by Day 17, culminating in animal deaths. [Figure 47] Figure 47. Red blood cell counts in rats repeatedly administered vehicle or ADC. [Figure 48] Figure 48. Neutrophil counts in rats repeatedly administered vehicle or ADC. [Figure 49] Figure 49. Reticulocyte counts in rats repeatedly administered vehicle or ADC. [Figure 50] Figure 50. Lymphocyte counts in rats repeatedly administered vehicle or ADC. [Figure 51] Figure 51. Platelet counts in rats repeatedly administered vehicle or ADC. [Figure 52] Figure 52. Alanine aminotransferase counts in rats repeatedly administered vehicle or ADC. [Figure 53] Figure 53. Aspartate aminotransferase counts in rats repeatedly administered vehicle or ADC. [Figure 54]Figure 54 shows a toxicokinetic analysis of rat plasma samples from repeat-dose non-GLP rat toxicity study #2. This analysis provides dose-level validation and demonstrates improved in vivo stability of the enfortumab compound 8 conjugate compared to the enfortumab vedotin conjugate. DETAILED DESCRIPTION OF THE INVENTION

[0022] definition "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. This term encompasses straight-chain and branched-chain hydrocarbyl groups, such as, by way of example, methyl (CH-), ethyl (CHCH-), n-propyl (CHCHCH-), isopropyl ((CH)CH-), n-butyl (CHCHCHCH-), isobutyl ((CH)CHCH-), sec-butyl ((CH)(CHCH)CH-), t-butyl ((CH)C-), n-pentyl (CHCHCHCHCHCH-), and neopentyl ((CH)CCH-).

[0023] The term "substituted alkyl" refers to an alkyl group, as defined herein, wherein one or more carbon atoms within the alkyl chain (excluding the C1 carbon atom) are optionally substituted with -O-, -N-, -S-, -S(O) n- (where n is 0-2), -NR- (where R is hydrogen or alkyl), and substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b wherein R' and R" may be the same or different and refer to alkyl groups selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

[0024] "Alkylene" preferably has 1 to 6, more preferably 1 to 3 carbon atoms, and is either straight-chain or branched, and optionally includes -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, by way of example, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), iso-propylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0025] "Substituted alkylene" refers to an alkylene group in which one to three hydrogens are replaced with a substituent as described with respect to carbon in the definition of "substituted" below.

[0026] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0027] The terms "alkylaminoalkyl," "alkylaminoalkenyl," and "alkylaminoalkynyl" refer to an R'NHR"- group where R' is an alkyl group, as defined herein, and R" is an alkylene, alkenylene, or alkynylene group, as defined herein.

[0028] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0029] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0030] The term "substituted alkoxy" refers to substituted alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where substituted alkyl, substituted alkenyl, cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0031] The term "alkoxyamino" refers to the group --NH-alkoxy, where alkoxy is as defined herein.

[0032] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group and includes groups such as trifluoromethoxy.

[0033] The term "haloalkyl" refers to a substituted alkyl group, as defined above, in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0034] The term "alkylalkoxy" refers to -alkylene-O-alkyl groups, alkylene-O-substituted alkyl groups, substituted alkylene-O-alkyl groups, and substituted alkylene-O-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0035] The term "alkylthioalkoxy" refers to -alkylene-S-alkyl groups, alkylene-S-substituted alkyl groups, substituted alkylene-S-alkyl groups, and substituted alkylene-S-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0036] "Alkenyl" refers to straight- or branched-chain hydrocarbyl groups having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1, and preferably 1 to 2, sites of double bond unsaturation. The term includes, by way of example, bivinyl, allyl, and but-3-en-1-yl. Cis- and trans-isomers, as well as mixtures of these isomers, are also encompassed by the term.

[0037] The term "substituted alkenyl" includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thiohetero ... "Alkenyl" refers to an alkenyl group as defined herein having 1 to 5 substituents, or 1 to 3 substituents selected from chlorooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0038] "Alkynyl" refers to a straight-chain or branched monovalent hydrocarbyl group having from 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, and preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0039] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0040] "Alkynyloxy" refers to an -O-alkynyl group, where alkynyl is as defined herein. Examples of alkynyloxy include ethynyloxy, propynyloxy, and the like.

[0041] "Acyl" refers to an HC(O)- group, an alkyl-C(O)- group, a substituted alkyl-C(O)- group, an alkenyl-C(O)- group, a substituted alkyl-C(O)- group, an alkenyl-C(O)- group, a substituted aryl-C(O)- group, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. "C(O)-" refers to substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)- groups. For example, acyl includes the "acetyl" group CH3C(O)-.

[0042] "Acylamino" is R 20 is hydrogen or alkyl, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein; —NR 20 C(O) alkyl group, -NR 20 C(O)-substituted alkyl group, NR 20 C(O)cycloalkyl group, -NR 20 C(O)-substituted cycloalkyl groups, -NR 20 C(O) cycloalkenyl group, -NR 20 C(O)-substituted cycloalkenyl group, -NR 20 C(O) alkenyl group, -NR 20 C(O) substituted alkenyl group, - NR 20 C(O) alkynyl group, -NR 20 C(O)-substituted alkynyl group, -NR 20 C(O) aryl group, -NR20 C(O) substituted aryl group, -NR 20 C(O) heteroaryl group, -NR 20 C(O)-substituted heteroaryl groups, -NR 20 C(O) heterocyclyl groups, and -NR 20 It refers to a C(O)-substituted heterocyclyl group.

[0043] The term "aminocarbonyl" or "aminoacyl" refers to R 21 and R 22 are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl; R 21 and R 22 optionally taken together with the nitrogen to which they are attached form a heterocyclyl or substituted heterocyclyl group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein, —C(O)NR 21 R 22 Refers to the base.

[0044] "Aminocarbonylamino" is R 21 , R 22 , and R 23 are each independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups together form a heterocyclyl group; 21 C(O)NR 22 R 23 Refers to the base.

[0045] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is, independently of the other, hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0046] The term "acyloxy" refers to alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- groups, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0047] "Aminosulfonyl" is R 21 and R 22 are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl; and optionally, R 21 and R 22 together with the nitrogen to which they are attached form a heterocyclyl or substituted heterocyclyl group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein; -SONR 21 R 22 Refers to the base.

[0048] "Sulfonylamino" is R 21 and R 22are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl; and optionally R 21 and R 22 But they are combined together with the atom(s) forming a heterocyclyl or substituted heterocyclyl group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein; -NR 21 SO2R 22 Refers to the base.

[0049] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as that found in a phenyl group) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring. The term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition of the aryl substituent, such aryl groups are optionally substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0050] "Aryloxy" refers to the group --O-aryl, where aryl is as defined herein, and including, but not limited to, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as defined herein.

[0051] "Amino" refers to the group -NH2.

[0052] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, with the proviso that at least one R is not hydrogen.

[0053] The term "azido" refers to the group -N3.

[0054] "Carboxyl", "carboxy", or "carboxylate" refers to -CO2H or its salts.

[0055] The term "carboxyl ester" or "carboxy esters", or "carboxyalkyl" or "carboxylalkyl" refers to a group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl, where alkyl, substituted alkyl, alkenyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein; -C(O)O-alkyl group, -C(O)O-substituted alkyl group, -C(O)O-alkenyl group, -C(O)O-substituted alkenyl group, -C(O)O-alkynyl group, -C(O)O-substituted alkynyl group, -C(O)O-aryl group, -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-heterocyclyl, and -C(O)O-substituted heterocyclyl groups.

[0056] "(Carboxyl ester)oxy" or "carbonate" refers to an -OC(O)O-alkyl group, an -OC(O)O-substituted alkyl group, an -OC(O)O-alkenyl ... -OC(O)O-substituted alkenyl groups, -OC(O)O-alkynyl groups, -OC(O)O-substituted alkynyl groups, -OC(O)O-aryl groups, -OC(O)O-substituted aryl groups, -OC(O)O-cycloalkyl groups, -OC(O)O-substituted cycloalkyl groups, -OC(O)O-cycloalkenyl groups, -OC(O)O-substituted cycloalkenyl groups, -OC(O)O-heteroaryl groups, -OC(O)O-substituted heteroaryl groups, -OC(O)O-heterocyclyl groups, and -OC(O)O-substituted heterocyclyl groups.

[0057] "Cyano" or "nitrile" refers to the group --CN.

[0058] "Cycloalkyl" refers to cyclic alkyl groups having one or more cyclic rings, including fused, bridged, and spiro ring systems, having 3 to 10 carbon atoms. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0059] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0060] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group having single or multiple rings and having at least one double bond, preferably 1 to 2 double bonds, and having 3 to 10 carbon atoms.

[0061] The term "substituted cycloalkenyl" has 1 to 5 substituents, or 1 to 3 substituents, and includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioa This refers to a cycloalkenyl group selected from alkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0062] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group having from 5 to 10 carbon atoms having single or multiple rings and at least one triple bond.

[0063] "Cycloalkoxy" refers to -O-cycloalkyl.

[0064] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0065] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0066] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0067] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms (e.g., 1 to 10 carbon atoms) and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or multiple fused rings within the ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), in which at least one ring within the ring system is aromatic. To satisfy valency requirements, any heteroatom within such a heteroaryl ring may be bonded or unbonded to an H or a substituent, such as an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of a heteroaryl group are optionally oxidized to provide an N-oxide group (N→O), a sulfinyl group, or a sulfonyl group. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of a heteroaryl substituent, such heteroaryl groups are optionally substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0068] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0069] "Heteroaryloxy" refers to -O-heteroaryl.

[0070] "Heterocycle", "heterocyclic", "heterocycloalkyl", and "heterocyclyl" are " refers to saturated or unsaturated groups having 3 to 20 ring atoms, including 1 to 10 heteroatoms, having a single ring or multiple fused rings, including fused ring systems, bridged ring systems, and spiro ring systems. These ring atoms are selected from nitrogen, sulfur, or oxygen; in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is not through the aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of a heterocyclyl group are optionally oxidized to provide an N-oxide group, an -S(O)- group, or an -SO2- group. To satisfy valency requirements, any heteroatom in such a heterocycle may be bound or unbound to one or more H or one or more substituents, such as alkyl groups or other substituents as described herein.

[0071] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, and 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, and the like.

[0072] Unless otherwise constrained by the definition of a heterocyclyl substituent, such heterocyclyl groups can be optionally substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0073] "Heterocyclyloxy" refers to the group --O-heterocyclyl.

[0074] The term "heterocyclylthio" refers to the group heterocyclyl-S-.

[0075] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0076] The term "hydroxyamino" refers to the group --NHOH.

[0077] "Nitro" refers to the -NO2 group.

[0078] "Oxo" refers to the atom (=O).

[0079] "Sulfonyl" refers to an S0-alkyl group, a S0-substituted alkyl group, a S0-alkenyl group, a S0-substituted alkenyl group, a S0-cycloalkyl group, a S0-substituted cycloalkyl group, a S0-cycloalkenyl group, a S0-substituted cycloalkenyl group, a S0-aryl group, a S0-substituted aryl group, a S0-heteroaryl group, a S0-substituted heteroaryl group, a S0-heterocyclyl group, and a S0-substituted heterocyclyl group, where S0-aryl ... by way of example. Sulfonyl includes, by way of example, methyl-S0-, phenyl-S0-, and 4-methylphenyl-S0-.

[0080] "Sulfonyloxy" refers to an -OSO2-alkyl group, an -OSO2-substituted alkyl group, an -OSO2-alkenyl group, an -OSO2-substituted alkenyl group, an -OSO2-cycloalkyl group, an -OSO2-substituted cycloalkyl group, an -OSO2-cycloalkenyl group, an -OSO2-substituted cycloalkenyl group, an -OSO2-aryl group, an -OSO2-substituted aryl group, an -OSO2-heteroaryl group, an -OSO2-substituted heteroaryl group, an -OSO2-heterocyclyl group, and an -OSO2-substituted heterocyclyl group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, OSO2-substituted cycloalkenyl group, an -OSO2-aryl group, an -OSO2-substituted aryl group, an -OSO2-heteroaryl group, an -OSO2-substituted heteroaryl group, an -OSO2-heterocyclyl group, and an -OSO2-substituted heterocyclyl group.

[0081] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is, independently of the other, hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0082] "Thiol" refers to the group --SH.

[0083] The term "thioxo" or "thioketo" refers to the (=S) atom.

[0084] The term "alkylthio" or "thioalkoxy" refers to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, the sulfur may be oxidized to -S(O)-. This sulfoxide may exist as one or more stereoisomers.

[0085] The term "substituted thioalkoxy" refers to the group --S-substituted alkyl.

[0086] The term "thioaryloxy" refers to an aryl-S- group, where the aryl group is as defined herein, including optionally substituted aryl groups, as defined herein.

[0087] The term "thioheteroaryloxy" refers to the group heteroaryl-S-, where the heteroaryl group is as defined herein and includes optionally substituted aryl groups, as defined herein.

[0088] The term "thioheterocyclooxy" refers to the group heterocyclyl-S-, in which the heterocyclyl group is as defined herein, including optionally substituted heterocyclyl groups, which are as defined herein.

[0089] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can also mean that one or more hydrogen atoms of the particular group or radical are each, independently of one another, replaced with the same or different substituents as defined below.

[0090] In addition to the groups disclosed for individual terms herein, one or more hydrogens on a saturated carbon atom of a particular group or radical (any two hydrogens on one carbon may be substituted, e.g., ═O, ═NR 70 , =N-OR 70 Substituents for replacing ═, ... 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M +, -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)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)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 and R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, and each R 70are, independently of each other, hydrogen or R 60 and each R 80 are, independently of each other, R 70 Or, two R 80’ are taken together with the nitrogen atom to which they are attached to form a 5-, 6-, or 7-membered heterocycloalkyl which may optionally contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, where N may have —H or C1-C3 alkyl substitution; each M + is a counterion with a net positive charge. + are independent of each other, e.g., K + , Na + , Li + Alkaline ions such as; + N(R 60 ) 4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The "0.5" subscript means that one of the counterions to such divalent alkaline earth ions may be an ionized form of a compound of the present invention and the other may be a typical counterion such as chloride, or that a two-ionized compound disclosed herein may serve as a counterion to such divalent alkaline earth ion, or that a doubly-ionized compound of the present invention may serve as a counterion to such divalent alkaline earth ion.) Specific examples include -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0091] Further to the disclosure herein, substituents for hydrogens on unsaturated carbon atoms of "substituted" alkene, alkyne, aryl, and heteroaryl groups are represented by -R unless otherwise specified.60 、ハロ、-O - M + 、-OR 70 、-SR 70 、-S - M + 、-NR 80 R 80 、トリハロメチル、-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 70C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 , and M + is as defined above, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0092] In addition to the groups disclosed for each individual term herein, substituents on the hydrogen on the nitrogen atom of "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 , and M + is as defined above.

[0093] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0094] For all of the substituents defined above, it should be understood that polymers derived from defining a substituent with further substituents thereon (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which in turn is substituted with a substituted aryl group) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0095] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is derived from the terminal portion of the functional group followed by the adjacent functional group, naming it toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0096] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the subject compounds encompass all stereochemical isomers arising from the substitution of the compounds.

[0097] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (a salt with counterions that are acceptable for mammalian safety in a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" can be derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium. "A" refers to a pharmaceutically acceptable salt of a compound, including ammonium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and, if the molecule contains a basic functional group, to a salt of an organic or inorganic acid, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0098] The term "salt thereof" refers to compounds formed when a hydrogen ion of an acid is replaced with a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds not intended for administration to a patient. Exemplary salts of the present compounds include those in which the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid being the anionic component of the salt.

[0099] "Solvate" refers to a complex formed by the combination of solvent molecules with solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture thereof. 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.

[0100] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atomic connectivity but different arrangements of the atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0101] "Tautomers" refers to alternating forms of molecules that differ only in the position of the atoms' electron bonding and / or hydrogen ions, such as enol-keto tautomers and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing the ring atom arrangement -N=C(H)-NH-, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are possible.

[0102] It will be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to encompass all permutations of salts, solvates, and stereoisomers, such as pharmaceutically acceptable salt solvates of stereoisomers of the subject compound.

[0103] The terms "antibody" and "immunoglobulin" encompass antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which in turn is composed of two dimers of a heavy and a light polypeptide chain); single-chain antibodies (e.g., scFv); fragments of antibodies (e.g., whole antibodies or single-chain antibody fragments) that retain specific binding to an antigen, including, but not limited to, Fab fragments, Fv fragments, scFv fragments, and Fd fragments; chimeric antibodies; humanized antibodies; single-chain antibodies; and fusion proteins comprising the antigen-binding portion of an antibody and a protein other than an antibody. These antibodies may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. These antibodies may be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair). These antibodies may be bound to a solid phase, including, but not limited to, a polystyrene plate or beads. Also encompassed by the term are Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to the antigen, as well as monoclonal antibodies. Antibodies may be monovalent or bivalent.

[0104] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, 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. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment (a name reflecting its ease of crystallization). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0105] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, these six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (i.e., half of an Fv, containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the complete binding site.

[0106] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab', in which the cysteine ​​residues of the constant domains display free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have the hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0107] The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Based on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM), some of which may be further divided into subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2).

[0108] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0109] The term "diabody" refers to a group of identical polypeptide chains (V H -V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) refers to a small antibody fragment having two antigen-binding sites. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites.

[0110] As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two agents, expressed as the dissociation constant (Kd). 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. The affinity of an antibody for a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein in reference to antibodies and / or antigen-binding fragments.

[0111] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic interactions, and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. The subject anti-Nectin-4 antibodies specifically bind to an epitope within a Nectin-4 polypeptide, e.g., a human Nectin-4 polypeptide, e.g., glycosylated Nectin-4 or a fragment thereof. Non-specific binding occurs when the affinity is less than about 10 -7 binding that is less than M, e.g., an affinity of 10 -6 M, 10 -5 M, 10 -4 M, etc.

[0112] The term "specifically binds" in the context of antibodies and antigens means, for example, about 10 5M -1 or greater affinity or K a (i.e., the equilibrium association constant of a particular binding interaction, expressed in units of 1 / M) refers to the rate at which an antibody binds to or associates with an antigen.

[0113] "High affinity" binding is defined as binding with a 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or higher K a Alternatively, affinity refers to the equilibrium dissociation constant (K) of a particular binding interaction, which has units of M. D ) (e.g., 10 -5 M~10 -13 In some embodiments, specific binding is defined as about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M or less, 10 -10 M, 10 -11 M or 10 -12 M or lower K D means that the antibody binds to the antigen. The binding affinity of an antibody to an antigen can be readily determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., using a BIAcore2000 instrument, using the general procedures outlined by the manufacturer); radioimmunoassay; and the like.

[0114] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the non-contiguous antigen-binding sites present in the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and Mac Callum et al., J. Mol. Biol. 262:732-745 (1996), whose definitions, when compared with each other, include overlapping or subsets of amino acid residues. However, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in Table 1 below for comparison.

[0115] [Table 1]

[0116] Throughout this disclosure, the numbering of residues within immunoglobulin heavy chains and immunoglobulin light chains follows the method described in MacCallum et al., which is expressly incorporated herein by reference. al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0117] As used herein, the term "framework" when used in reference to an antibody variable region is intended to refer to all amino acid residues outside the CDR regions within the variable region of an antibody. The variable region framework is generally a discontinuous amino acid sequence about 100 to 120 amino acids in length, but is intended to refer only to the amino acids outside the CDRs. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs.

[0118] A "native Ig polypeptide" is a polypeptide comprising an amino acid sequence that does not have an aldehyde-tagged constant region as described herein. A native polypeptide may comprise a native-sequence constant region or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions).

[0119] In the context of Ig polypeptides, the term "constant region" is well understood in the art and refers to the C-terminal region of an Ig heavy chain or an Ig light chain. The Ig heavy chain constant region comprises a CH1 domain, a CH2 domain, and a CH3 domain (and a CH4 domain, if the heavy chain is a μ or ε heavy chain). In native Ig heavy chains, the CH1 domain, CH2 domain, CH3 domain (and, if present, the CH4 domain) begin immediately (C-terminally) after the heavy chain variable (VH) region and are each about 100 amino acids long. In a native Ig light chain, the constant region begins immediately (C-terminal side) after the light chain variable (VL) region and is approximately 100 to 120 amino acids long.

[0120] An "epitope" is a site on an antigen (e.g., a site on Nectin-4) to which an antibody binds. Epitopes can be formed from either contiguous amino acids or noncontiguous amino acids that are adjacent due to protein folding (e.g., tertiary folding). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three amino acids, more often at least five or eight to ten amino acids, in a linear or spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Several commercial laboratories offer epitope mapping services. Epitopes bound by antibodies that are immunoreactive with membrane-associated antigens may be present on the surface of the cell (e.g., within the extracellular region of a transmembrane protein), and therefore such epitopes are considered to be cell-surface-exposed, solvent-exposed, and / or cell-surface-exposed.

[0121] "Genetically encodable," when used in reference to an amino acid sequence of a polypeptide, peptide, or protein, means that the amino acid sequence is composed of amino acid residues that can be produced by transcription and translation of a nucleic acid encoding the amino acid sequence, where transcription and / or translation can occur in a cell or in a cell-free in vitro transcription / translation system.

[0122] The term "control sequences" refers to DNA sequences that facilitate the expression of an operably linked coding sequence in a particular expression system, such as mammalian cells, bacterial cells, cell-free synthesis, etc. Control sequences that are suitable for prokaryotic systems include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic systems may utilize promoters, polyadenylation signals, and enhancers.

[0123] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to DNA for a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation initiation. Typically, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking is accomplished by ligation or through an amplification reaction. Conventionally, synthetic oligonucleotide adapters or linkers can be used to link sequences.

[0124] The term "expression cassette," as used herein, refers to a nucleic acid segment, usually a DNA segment, that can be inserted into a nucleic acid (e.g., using restriction enzyme recognition sites compatible for ligation into a construct of interest or by homologous recombination into the construct of interest or into the host cell genome). Typically, the nucleic acid segment comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction enzyme recognition sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. Expression cassettes are used to express a polypeptide of interest in a host cell, e.g., a mammalian host cell. The gene may also contain elements that facilitate expression of a polynucleotide encoding the gene. These elements may include, but are not limited to, a promoter, a minimal promoter, an enhancer, a response element, a transcription termination sequence, a polyadenylation sequence, and the like.

[0125] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include proteinaceous or nonproteinaceous solutes, such as enzymes, hormones, and the like. In some embodiments, the antibody is purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99% by weight, based on the antibody, as measured by the Lowry assay; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions with Coomassie blue or silver staining. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In some cases, isolated antibody is prepared by at least one purification step.

[0126] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are produced 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 antibody-producing cells isolated from a first animal immunized with an antigen is a natural antibody.

[0127] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody containing one or more amino acids (e.g., in the framework region, constant region, or CDR) substituted with an amino acid present at the corresponding position from a human antibody. Typically, a humanized antibody results in a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting, veneering or resurfacing, chain shuffling, and the like. In certain embodiments, framework substitutions are identified by modeling the interactions of CDR 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.

[0128] In some embodiments, an antibody molecule disclosed herein comprises a heavy chain comprising a heavy chain variable region as provided herein and a human IgG1 constant region having the amino acid sequence set forth in UniProt:P01857-1, version 1. In some embodiments, an antibody molecule disclosed herein comprises a light chain comprising a light chain variable region as provided herein and a human light chain constant region. In some embodiments, the human light chain constant region comprises a human kappa light chain constant region having the amino acid sequence set forth in UniProtKB / Swiss-Prot:P01834.2. In some embodiments, the human IgG1 heavy chain constant region present in a subject antibody may comprise mutations, such as substitutions, to modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody-dependent cellular cytotoxicity (ADCC). In some cases, only the L234A and L235A mutations are used, without the P329G mutation. The numbering of these substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is a It is commonly used when referring to residues within the globulin heavy chain constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0129] The term "chimeric antibody" refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, variable region gene segments from a mouse monoclonal antibody may be joined to human constant region gene segments, such as γ1 and γ3. An example of a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, although domains from other mammalian species may also be used.

[0130] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acids of any length in polymeric form. Unless specifically indicated otherwise, "polypeptide," "peptide," and "protein" can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term encompasses fusion proteins, including, but not limited to, fusion proteins with 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); immunologically tagged proteins; and the like. In the context of antibodies, it is clear that a chain or domain includes a polypeptide.

[0131] "Native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide before it is modified to contain a modified amino acid residue.

[0132] The terms "amino acid analog," "unnatural amino acid," and the like, may be used interchangeably and include amino acid-like compounds similar in structure and / or overall shape to one or more amino acids normally found in natural proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as the naturally occurring D-form, as well as L-form amino acid analogs. In some cases, amino acid analogs share the backbone and / or side chain structure of one or more naturally occurring amino acids, differing in one or more modified groups in the molecule. Such modifications can include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as a methyl group or a hydroxyl group) or atom (such as Cl or Br), deletion of a group, replacement of a covalent bond (such as replacement of a single bond with a double bond), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids, α-amino acids, and the like.

[0133] The terms "amino acid side chain" or "side chain of an amino acid" and the like refer to naturally occurring amino acids, non-naturally occurring amino acids, and the like. The term "amino acid side chain" may also be used to refer to a substituent attached to the alpha carbon of an amino acid residue, including amino acids and amino acid analogs. Amino acid side chains may also include amino acid side chains as described in the context of modified amino acids and / or conjugates described herein.

[0134] The term "conjugated" generally refers to a covalent or non-covalent, usually covalent, chemical bond that proximally links one molecule of interest with a second molecule of interest. In some embodiments, the agent is selected from a half-life extending group, a labeling agent, and a therapeutic agent. For example, in the case of half-life extension, the antibody of the present disclosure may optionally be modified (e.g., by pegylation, hyperglycosylation, etc.) to improve the pharmacokinetic profile. Of interest are modifications that can extend serum half-life.

[0135] Terms such as "glycan" may be used to refer to monosaccharide, disaccharide, oligosaccharide, and polysaccharide monomers and / or polymers. The term sugar may be used to refer to smaller glycans, such as monosaccharides and disaccharides. The term "glycan derivative" encompasses compounds in which one or more functional groups of the subject glycan have been substituted (replaced with any convenient substituent), modified (converted to another group using any convenient chemistry), or absent (e.g., removed or replaced with H). A variety of glycans and glycan derivatives are available and can be adapted for use in the subject compounds and conjugates.

[0136] As used herein, the term "isolated" is intended to describe a compound of interest that is in an environment that is different from the environment that the compound naturally occurs in. "Isolated" is intended to include the compound being present in a sample in which the compound of interest has been significantly enriched and / or in which the compound of interest has been partially or substantially purified.

[0137] As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free from other components that are naturally associated with it.

[0138] The term "physiological conditions" is intended to encompass conditions compatible with living cells, eg, primarily aqueous conditions in which temperature, pH, salinity, etc. are compatible with living cells.

[0139] "Reactive partner" refers to a molecule or molecular substructure that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include a cysteine ​​or serine of a sulfatase motif with a formylglycine generating enzyme (FGE), which react to form a reaction product, a converted aldehyde tag, that contains formylglycine (fGly) in place of the cysteine ​​or serine in the motif. Other exemplary reactive partners include an aldehyde (e.g., reactive aldehyde group) of the fGly residue of a converted aldehyde tag, and an "aldehyde-reactive reactive partner" that contains an aldehyde-reactive group and a moiety of interest, which react to form a reaction product, a polypeptide having a moiety of interest conjugated to the polypeptide via the fGly residue.

[0140] "N-terminus" refers to the terminal amino acid residue of a polypeptide having a free amino group; the amino group of the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0141] "C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group; the carboxyl group of the non-C-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0142] "Internal site," when used in reference to a polypeptide or the amino acid sequence of a polypeptide, means a region of the polypeptide that is neither the N-terminus nor the C-terminus.

[0143] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. This effect may be preventative, in terms of a complete or partial suppression of a disease or its symptoms, and / or therapeutic, in terms of a partial or complete cure of a disease and / or adverse effects caused by the disease. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject susceptible to the disease but not yet diagnosed as having the disease; (b) inhibiting the disease, e.g., arresting its occurrence; and (c) relieving the disease, e.g., causing regression of the disease.

[0144] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0145] A "therapeutically effective amount" or "effective amount" refers to the amount of a subject anti-Nectin-4 antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to treat such condition. The "therapeutically effective amount" will vary depending on the anti-Nectin-4 antibody, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0146] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. Also, the scope of the present invention is defined only by the appended claims, and it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0147] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value within that stated range, is also encompassed within the invention. Where there are specifically excluded limits in the stated range, the upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0149] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. Please note that the terms "and" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of such antibodies, a reference to a "CDR" refers to one or more CDRs and equivalents thereof known to those skilled in the art, and so forth. Furthermore, please note that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a guide prior to use of exclusive terminology such as "solely," "only," or the use of "negative" limitations in connection with the recitation of claim elements.

[0150] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may require independent confirmation.

[0151] Detailed Description The present disclosure provides antibodies specific to Nectin-4. The present disclosure also provides antibody conjugates, e.g., ADCs, comprising such Nectin-4-specific antibodies. The present disclosure provides methods for producing such antibodies and conjugates, as well as methods for using such antibodies and conjugates. Each embodiment is described in more detail in the following sections. Compositions comprising the antibodies and / or ADCs of the present disclosure are also provided, and in some cases, include pharmaceutical compositions. In one aspect, a method of using an ADC of the present disclosure is provided, comprising administering a therapeutically effective amount of the ADC to an individual with a cell proliferative disorder.

[0152] Nectin-4 antibody and its antibody-drug conjugate As summarized above, the present disclosure provides antibodies specific to Nectin-4 and conjugates of such antibodies (e.g., antibody-drug conjugates (ADCs)). In addition, the present disclosure provides anti-Nectin-4 antibodies that contain fGly residues.

[0153] antibody-drug conjugates The present disclosure provides conjugates of antibodies specific to Nectin-4, e.g., ADCs. By "conjugate" is meant a polypeptide (e.g., an antibody) covalently bound to a moiety of interest (e.g., a drug or active agent). For example, antibody-drug conjugates according to the present disclosure include those in which one or more drugs or active agents are covalently bound to an antibody. In some embodiments, the polypeptide (e.g., an antibody) and the one or more drugs or active agents are bound to each other via one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds may include a linker, such as a cleavable linker, as described herein.

[0154] In some embodiments, the complex is a polypeptide complex comprising a polypeptide (e.g., an antibody) conjugated to one or more other substructures. In some embodiments, the one or more substructures conjugated to the polypeptide can be, independently of one another, any of a variety of substructures of interest, including, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a substructure for immobilizing the polypeptide on a membrane or surface. In some embodiments, the complex is a drug conjugate in which the polypeptide is an antibody, i.e., an antibody-drug conjugate is provided. For example, the complex can be a drug conjugate in which the polypeptide is conjugated to one or more drugs or active agents. Various types of drugs and active agents may be used in the complex, as described in more detail below.

[0155] One or more drugs or active agents may be conjugated to a polypeptide (e.g., an antibody) at any desired site on the polypeptide. provides, for example, polypeptides having one or more drugs or active agents conjugated at or near the C-terminus of the polypeptide. Other examples include polypeptides having one or more drugs or active agents conjugated at or near the N-terminus of the polypeptide. Also included are polypeptides having one or more drugs or active agents conjugated between the C-terminus and N-terminus of the polypeptide (e.g., at an internal site in the polypeptide). Combinations of the above are also possible, in which a polypeptide is conjugated to more than one drug or active agent.

[0156] In certain embodiments, conjugates of the present disclosure include those in which one or more drugs or active agents are conjugated to an amino acid residue of a polypeptide at the alpha carbon of the amino acid residue. Stated another way, a conjugate comprises a polypeptide in which the side chains of one or more amino acid residues within the polypeptide have been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents via a linker as described herein). For example, a conjugate comprises a polypeptide in which the alpha carbon of one or more amino acid residues within the polypeptide have been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents via a linker as described herein).

[0157] Embodiments of the present disclosure encompass complexes in which a polypeptide is conjugated to one or more substructures, such as two, three, four, five, six, seven, eight, nine, or ten substructures, or more. The substructures may be conjugated to the polypeptide at one or more sites within the polypeptide. For example, one or more substructures may be conjugated to a single amino acid residue of the polypeptide. In some cases, one substructure is conjugated to an amino acid residue of the polypeptide. In other embodiments, two substructures may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first substructure is conjugated to a first amino acid residue of the polypeptide and a second substructure is conjugated to a second amino acid residue of the polypeptide. In other embodiments, two substructures may be conjugated to a first amino acid residue of the polypeptide and two substructures may be conjugated to a second amino acid residue of the polypeptide. For example, combinations of the above are possible where a polypeptide is conjugated to a first substructure at a first amino acid residue and to two other substructures at a second amino acid residue. Other combinations are also possible, including, but not limited to, a polypeptide conjugated to a first substructure and a second substructure at a first amino acid residue and to a third substructure and a fourth substructure at a second amino acid residue.

[0158] One or more amino acid residues of a polypeptide conjugated to one or more substructures of interest may be natural amino acids, unnatural amino acids, or a combination thereof. For example, a complex may include one or more drugs or active agents conjugated to natural amino acid residues of the polypeptide. In other cases, a complex may include one or more drugs or active agents conjugated to unnatural amino acid residues of the polypeptide. One or more drugs or active agents may be conjugated to the polypeptide at one natural amino acid residue or unnatural amino acid residue as described herein. One or more natural amino acid residues or unnatural amino acid residues within a polypeptide may be conjugated to one or more substructures of interest as described herein. For example, two (or more) amino acid residues (e.g., natural amino acid residues or unnatural amino acid residues) within a polypeptide may each be conjugated to one or more substructures of interest, such that multiple sites within the polypeptide are conjugated to one or more substructures.

[0159] In some embodiments, a polypeptide (e.g., an antibody) and a target substructure (e.g., a drug or active agent) are conjugated via a conjugation moiety. For example, the polypeptide and the target substructure may each be bound (e.g., covalently bound) to a conjugation moiety, i.e., the polypeptide and the target substructure may be indirectly linked via the conjugation moiety. In some cases, the conjugation moiety includes a hydrazinyl-indolyl compound or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl compound or a hydrazinyl-pyrrolo-pyridinyl compound. For example, a general scheme for linking a target substructure to a polypeptide via a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the following general reaction scheme: Hydrazinyl-indolyl conjugation moieties and hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also referred to herein as hydrazino-iso-Pictet-Spengler (HIPS) conjugation moieties and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugation moieties, respectively.

[0160] [ka]

[0161] In the above reaction scheme, each R independently comprises a moiety of interest (e.g., a drug or active agent) conjugated to a polypeptide (e.g., conjugated to a polypeptide via a linker as described herein), and n is an integer from 0 to 4. As shown in the above reaction scheme, a conjugation moiety (e.g., a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety) is attached to one or more drugs or active agents R. A polypeptide (e.g., an antibody) comprising a 2-formylglycine residue (fGly) reacts with the conjugation moiety to generate a polypeptide (e.g., antibody) conjugate, thereby attaching one or more drugs or active agents to the polypeptide via the conjugation moiety.

[0162] As described herein, the moiety of interest (also referred to herein as a "payload") can be any of a variety of moieties, including, but not limited to, a chemical entity such as a detectable label, or a drug or active agent. R' and R" can each, independently of the other, be any desired substituent, including, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z is CR 21 , N.R. 22 , which may be N, O, or S, and R 21 and R 22 are each independently selected from any of the substituents described for R' and R" above.

[0163] As shown in the conjugates and compounds described herein, other hydrazinyl-indolyl conjugation moieties or hydrazinyl-pyrrolo-pyridinyl conjugation moieties may be used. Linker moieties are also possible. For example, a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be attached (e.g., covalently attached) to one or more linkers. Thus, embodiments of the present disclosure include a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to one or more drugs or active agents via corresponding linkers. That is, a conjugate of the present disclosure may include one or more linkers, each linker attaching one or more corresponding drugs or active agents to the hydrazinyl-indolyl conjugation moiety or the hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety and one or more linkers may be collectively considered a "branched linker," in which the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is attached to two or more "branches," each of which comprises a linker attached to a drug or active agent.

[0164] For example, in some cases of the above reaction schemes, n is 0, so one R group (e.g., a drug or active agent) is attached to the hydrazinyl-indolyl conjugation moiety or the hydrazinyl-pyrrolo-pyridinyl conjugation moiety via a linker. In other cases, n is 1, so two R groups (e.g., a drug or active agent) are attached to the hydrazinyl-indolyl conjugation moiety or the hydrazinyl-pyrrolo-pyridinyl conjugation moiety, each via their own corresponding linker. In these cases, the hydrazinyl-indolyl conjugation moiety or the hydrazinyl-pyrrolo-pyridinyl conjugation moiety and the two linkers may be considered as a branched linker as a whole.

[0165] A combination of the same or different payloads may be conjugated to a polypeptide via a branched linker.In some embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are the same payload (e.g., drugs, active agents, or detectable labels).For example, the first branch of the branched linker may be attached to a payload (e.g., drugs, active agents, or detectable labels), and the second branch of the branched linker may be attached to the same payload (e.g., drugs, active agents, or detectable labels) as the first branch.

[0166] In other embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are different payloads (e.g., drugs, active agents, or detectable labels). For example, a first branch of the branched linker may be attached to a first payload (e.g., a first drug, active agent, or detectable label), and a second branch of the branched linker may be attached to a second payload (e.g., a second drug, active agent, or detectable label) that is different from the first payload (e.g., the first drug, active agent, or detectable label) attached to the first branch.

[0167] Various embodiments of linkers that can connect a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, such as a cleavable linker as described herein.

[0168] In certain embodiments, the polypeptide may be conjugated to one or more substructures of interest, where one or more amino acids of the polypeptide are modified prior to conjugation to one or more substructures of interest. Modification of amino acids can generate polypeptides containing one or more reactive groups suitable for conjugation to one or more substructures of interest. In some cases, a polypeptide can contain one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to one or more substructures of interest. For example, an amino acid of a polypeptide can be modified to contain a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be contained in an "aldehyde tag" or "ald-tag," which, as used herein, refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR) that has been converted to contain a 2-formylglycine residue (referred to as "fGly") by the action of formylglycine generating enzyme (FGE). The fGly residue generated by FGE is sometimes referred to as "formylglycine." In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence containing a "converted" sulfatase motif (i.e., a sulfatase motif in which the cysteine ​​or serine residue has been converted to fGly by the action of FGE, e.g., L(fGly)TPSR (SEQ ID NO: 245)). A converted sulfatase motif can be generated from an amino acid sequence containing an "unconverted" sulfatase motif (i.e., a sulfatase motif in which the cysteine ​​or serine residue has not been converted to fGly by FGE, but conversion is possible, e.g., an unconverted sulfatase motif having the sequence L(C / S)TPSR). "Conversion," when used in the context of the action of formylglycine generating enzyme (FGE) on a sulfatase motif, refers to the biochemical modification of a cysteine ​​or serine residue within the sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly or Ser to fGly). Further aspects of aldehyde tags and their use in site-specific protein modification are described in US Pat. Nos. 7,985,783 and 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0169] In some cases, to produce a conjugate, a polypeptide containing an fGly residue can be conjugated to one or more moieties of interest by reacting the fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above). For example, the fGly-containing polypeptide can be contacted with a reactive partner under conditions suitable for conjugating one or more drugs or active agents to the polypeptide. In some cases, the reactive partner can include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above. For example, one or more drugs or active agents can be attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, one or more drugs or active agents are attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, e.g., covalently attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, where each drug or active agent is attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety via a corresponding linker.

[0170] In certain embodiments, the conjugates of the present disclosure comprise a polypeptide (e.g., an antibody) having at least one amino acid residue attached to one or more moieties of interest (e.g., one or more drugs or active agents). To create the conjugate, the amino acid residues of the polypeptide may be modified and then linked to one or more drugs or active agents attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above. In certain embodiments, the polypeptide (e.g., an antibody) ) is a cysteine ​​or serine residue that is converted to an fGly residue as described above. In certain embodiments, the converted amino acid residue (e.g., an fGly residue) is conjugated to one or more drugs or active agents containing a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above, thereby providing a conjugate of the disclosure in which one or more drugs or active agents are conjugated to the polypeptide via a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As used herein, the term fGly' refers to an amino acid residue of a polypeptide (e.g., an antibody) linked to one or more moieties of interest (e.g., one or more drugs or active agents).

[0171] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one amino acid residue conjugated to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described herein, which in turn is conjugated to one or more drugs or active agents via one or more corresponding linkers. For example, the conjugate may comprise a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') conjugated to one or more moieties of interest (e.g., one or more drugs or active agents) as described above.

[0172] Embodiments of the present disclosure include conjugates of formula (I):

[0173] [ka] (I)

[0174] During the ceremony, Z 1 , Z 2 , Z 3 , and Z 4 are each, independently of each other,4 , N, and C.L. B -W 2 Selected from; R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each, independently of one another, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally joined in a ring to form a 5- or 6-membered heterocyclyl; Each R 4 are each independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl selected from acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker; L B is a second linker; W 1 is the first drug; W 2 is the second drug; W 3 is a polypeptide.

[0175] The substituents associated with the conjugates of formula (I) are described in more detail below.

[0176] In one embodiment, Z 1 , Z 2 , Z 3 , and Z 4 are each, independently of each other, 4 , N, and C.L. B -W 2 In one embodiment, Z 1 is CR 4 In one embodiment, Z 1 is N. In one embodiment, Z 1 is CL B -W 2 In one embodiment, Z 2 is CR 4 In one embodiment, Z 2 is N. In one embodiment, Z 2 is CL B -W 2 In one embodiment, Z 3 is CR 4 In one embodiment, Z 3 is N. In one embodiment, Z 3 is CL B -W 2 In one embodiment, Z 4 is CR 4 In one embodiment, Z 4 is N. In one embodiment, Z 4 is CL B -W 2 is.

[0177] Various Z 1 , Z 2 , Z 3 , and Z 4 For example, in some cases, Z 1 is CR 4 and Z 2 is CR4 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is N and Z 2 is CR 4 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is CL B -W 2 and Z 2 is CR 4 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CL B -W 2 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CR 4 and Z 3 is CL B -W 2 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CR 4 and Z 3 is CR 4 and Z 4 is CL B -W 2 is.

[0178] In one embodiment, R 1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 1 is hydrogen. In some embodiments, R 1 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 1 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 1 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkynyl or substituted alkynyl, such as a substituted alkenyl. 1 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 1 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 1 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Shik alkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 1 is heterocyclyl or substituted heterocyclyl, e.g., C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0179] In one embodiment, R 2 and R 3 are each, independently of one another, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally joined in a ring to form a 5- or 6-membered heterocyclyl.

[0180] In one embodiment, R 2is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 2 is hydrogen. In some embodiments, R 2 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 2 is methyl. In one embodiment, R 2 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 2 is alkynyl or substituted alkynyl. In some embodiments, R 2 is alkoxy or substituted alkoxy. In some embodiments, R 2 is amino or substituted amino. In one embodiment, R 2 is carboxyl or carboxyl ester. In one embodiment, R 2 is acyl or acyloxy. In one embodiment, R 2 is acylamino or aminoacyl. In one embodiment, R 2 is an alkylamide or a substituted alkylamide. In one embodiment, R 2is sulfonyl. In one embodiment, R 2 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 2 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 2 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 2 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 2 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0181] In one embodiment, R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkene In certain embodiments, R is selected from nyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 3 is hydrogen. In some embodiments, R 3 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 3 is methyl. In one embodiment, R 3 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 3 is alkynyl or substituted alkynyl. In some embodiments, R 3 is alkoxy or substituted alkoxy. In some embodiments, R 3 is amino or substituted amino. In one embodiment, R 3 is carboxyl or carboxyl ester. In one embodiment, R 3 is acyl or acyloxy. In one embodiment, R 3 is acylamino or aminoacyl. In one embodiment, R 3 is an alkylamide or a substituted alkylamide. In one embodiment, R 3 is sulfonyl. In one embodiment, R 3is thioalkoxy or substituted thioalkoxy. In some embodiments, R 3 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 3 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 3 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 3 Heterocyclyl or substituted heterocyclyl, for example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0182] In one embodiment, R 2 and R 3 are both methyl.

[0183] In one embodiment, R 2 and R 3 are optionally joined in a ring to form a 5- or 6-membered heterocyclyl. 2 and R 3are joined in a ring to form a 5- or 6-membered heterocyclyl. 2 and R 3 are linked together to form a 5-membered heterocyclyl. 2 and R 3 are joined together to form a six-membered heterocyclyl.

[0184] In one embodiment, each R 4 are each independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0185] Each R 4 The various possibilities for R are described in more detail below. In one embodiment, R 4 is hydrogen. In one embodiment, each R 4 is hydrogen. In some embodiments, R 4 is a halogen, such as F, Cl, Br, or I. In some embodiments, R 4 is F. In one embodiment, R 4 is Cl. In some embodiments, R 4 is Br. In one embodiment, R 4 is I. In one embodiment, R 4 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R4 is methyl. In one embodiment, R 4 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 4 is alkynyl or substituted alkynyl. In some embodiments, R 4 is alkoxy or substituted alkoxy. In some embodiments, R 4 is amino or substituted amino. In one embodiment, R 4 is carboxyl or carboxyl ester. In one embodiment, R 4 is acyl or acyloxy. In one embodiment, R 4 is acylamino or aminoacyl. In one embodiment, R 4 is an alkylamide or a substituted alkylamide. In one embodiment, R 4 is sulfonyl. In one embodiment, R 4 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 4 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 In some embodiments, R is a substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl (e.g., phenyl or substituted phenyl). 4 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 4 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 4 Heterocyclyl or substituted heterocyclyl, for example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0186] In one embodiment, L A is the first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0187] In one embodiment, 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.

[0188] In one embodiment, W 1 is the first drug (or first active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0189] In one embodiment, W 2 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.

[0190] In one embodiment, W 3 is a polypeptide (e.g., an antibody). 3comprises one or more fGly' residues as described herein. In certain embodiments, the polypeptide is linked to the remainder of the conjugate through an fGly' residue as described herein. Exemplary polypeptides and antibodies that can be used in the conjugates of the present disclosure are described in more detail below.

[0191] In certain embodiments, the conjugate of Formula (I) comprises a first linker, L A The first linker L A The first moiety of interest (e.g., a first drug or active agent) is attached to the conjugate. The first linker L may be used to link the first linker L to a polypeptide (e.g., an antibody) via a linking moiety. A may be attached (e.g., covalently attached) to the conjugation moiety (e.g., as described herein). For example, the first linker L A may attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a first drug or active agent. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be attached to the first linker L A (i.e., a first or active agent) to a polypeptide, such as an antibody.

[0192] For example, as shown in formula (I) above, L A via the conjugation moiety, W 3 is connected to W 3 is linked to the linker L via a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety A As mentioned above, W 3 is a polypeptide (e.g., an antibody), i.e., L Ais attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, e.g., a linker L A is indirectly attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0193] Any convenient linker may be used as the first linker, L, in the subject conjugates and compounds. A In one embodiment, the first linker L A may comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker L A may comprise an alkyl group or a substituted alkyl group. In one embodiment, the first linker L A may comprise an alkenyl group or a substituted alkenyl group. In certain embodiments, the first linker, L A may comprise an alkynyl group or a substituted alkynyl group. In one embodiment, the first linker, L A may comprise an alkoxy group or a substituted alkoxy group. In one embodiment, the first linker L A may comprise an amino or substituted amino group. In one embodiment, the first linker L A may contain a carboxyl group or a carboxyl ester group. In one embodiment, the first linker L A may comprise an acylamino group. In one embodiment, the first linker L A may comprise an alkylamide group or a substituted alkylamide group. In one embodiment, the first linker, L Amay comprise an aryl group or a substituted aryl group. In some embodiments, the first linker, L A may comprise a heteroaryl group or a substituted heteroaryl group. In some embodiments, the first linker, L A may comprise a cycloalkyl group or a substituted cycloalkyl group. In some embodiments, the first linker, L A may include a heterocyclyl group or a substituted heterocyclyl group.

[0194] In one embodiment, the first linker L A The polymer may comprise a polymer. For example, the polymer may comprise polyalkylene glycols and derivatives thereof, such as polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., these homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In some embodiments, the polymer is a polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol. Illustrated in the conjugates and compounds described in more detail below are: Other linkers are possible, such as those shown.

[0195] In some embodiments, L A is a first linker described by the formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, In the formula, L 1 , L 2 , L 3 , L4 , L 5 , and L 6 are each, independently of one another, a linker subunit; and a, b, c, d, e, and f are each, independently of one another, 0 or 1.

[0196] In some embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In some embodiments, the sum of a, b, c, d, e, and f is 0. In some embodiments, the sum of a, b, c, d, e, and f is 1. In some embodiments, the sum of a, b, c, d, e, and f is 2. In some embodiments, the sum of a, b, c, d, e, and f is 3. In some embodiments, the sum of a, b, c, d, e, and f is 4. In some embodiments, the sum of a, b, c, d, e, and f is 5. In some embodiments, the sum of a, b, c, d, e, and f is 6. In some embodiments, a, b, c, d, e, and f are each 1. In some embodiments, a, b, c, d, and e are each 1, and f is 0. In some embodiments, a, b, c, and d are each 1, and e and f are each 0. In some embodiments, a, b, and c are each 1, and d, e, and f are each 0. In some embodiments, a and b are each 1, and c, d, e, and f are each 0. In some embodiments, a is 1, and b, c, d, e, and f are each 0.

[0197] In one embodiment, the linker subunit L 1 is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 2 is the first drug or active agent W, if present 1 In one embodiment, the linker subunit L 3 is the first drug or active agent W, if present 1 In one embodiment, the linker subunit L 4is the first drug or active agent W, if present 1 In one embodiment, the linker subunit L 5 is the first drug or active agent W, if present 1 In one embodiment, the linker subunit L 6 is the first drug or active agent W, if present 1 is connected to

[0198] Any convenient linker subunit may be used as the first linker L A Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene, and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclyl groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 each (if present) independently comprises one or more groups 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 comprising alkylenediamines).

[0199] In some embodiments, L 1 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 1 comprises polyethylene glycol. In some embodiments, L 1 comprises a modified polyethylene glycol. In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 In some embodiments, L1 In some embodiments, L comprises an aryl group or a substituted aryl group. 1 is a diamine (e.g., alkylene diamine) amine-containing linking group).

[0200] In some embodiments, L 2 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 2 comprises polyethylene glycol. In some embodiments, L 2 comprises a modified polyethylene glycol. In some embodiments, L 2 comprises an amino acid residue. In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L comprises an aryl group or a substituted aryl group. 2 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0201] In some embodiments, L 3 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 3 comprises polyethylene glycol. In some embodiments, L 3 comprises a modified polyethylene glycol. In some embodiments, L 3 comprises an amino acid residue. In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L comprises an aryl group or a substituted aryl group. 3 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0202] In some embodiments, L 4(when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 comprises polyethylene glycol. In some embodiments, L 4 comprises a modified polyethylene glycol. In some embodiments, L 4 comprises an amino acid residue. In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L comprises an aryl group or a substituted aryl group. 4 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0203] In some embodiments, L 5 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 comprises polyethylene glycol. In some embodiments, L 5 comprises a modified polyethylene glycol. In some embodiments, L 5 comprises an amino acid residue. In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L comprises an aryl group or a substituted aryl group. 5 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0204] In some embodiments, L 6 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 6 comprises polyethylene glycol. In some embodiments, L 6comprises a modified polyethylene glycol. In some embodiments, L 6 comprises an amino acid residue. In some embodiments, L 6 In some embodiments, L 6 In some embodiments, L comprises an aryl group or a substituted aryl group. 6 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0205] In some embodiments, L A is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - a first linker comprising -(L 1 ) a -ha-(T 1 -V 1 ) a - and; -(L 2 ) b -ha-(T 2 -V 2 ) b - and; -(L 3 ) c -ha-(T 3 -V 3 ) c - and; -(L 4 ) d -ha-(T 4 -V 4 ) d - and; -(L 5 ) e -ha-(T 5 -V 5 ) e - and; -(L 6 ) f -ha-(T 6 -V6 ) f - and T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 If present, group); V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is a covalent bond or linking functional group, if present; a, b, c, d, e, and f are each independently 0 or 1.

[0206] In some embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In some embodiments, the sum of a, b, c, d, e, and f is 0. In some embodiments, the sum of a, b, c, d, e, and f is 1. In some embodiments, the sum of a, b, c, d, e, and f is 2. In some embodiments, the sum of a, b, c, d, e, and f is 3. In some embodiments, the sum of a, b, c, d, e, and f is 4. In some embodiments, the sum of a, b, c, d, e, and f is 5. In some embodiments, the sum of a, b, c, d, e, and f is 6. In some embodiments, a, b, c, d, e, and f are each 1. In some embodiments, a, b, c, d, and e are each 1, and f is 0. In some embodiments, a, b, c, and d are each 1, and e and f are each 0. In some embodiments, a, b, and c are each 1, and d, e, and f are each 0. In some embodiments, a and b are each 1, and c, d, e, and f are each 0. In some embodiments, a is 1, and b, c, d, e, and f are each 0.

[0207] As noted above, in one embodiment, L 1is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 1 is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, V 1 is attached to a first drug or active agent. In one embodiment, L 2 When present, T is attached to the first drug or active agent. 2 is attached to the first drug or active agent, if present, or V 2 When present, L is attached to the first drug or active agent. 3 When present, T is attached to the first drug or active agent. 3 is attached to the first drug or active agent, if present, or V 3 When present, L is attached to the first drug or active agent. 4 When present, T is attached to the first drug or active agent. 4 is attached to the first drug or active agent, if present, or V 4 When present, L is attached to the first drug or active agent. 5 When present, T is attached to the first drug or active agent. 5 is attached to the first drug or active agent, if present, or V 5 When present, L is attached to the first drug or active agent. 6 When present, T is attached to the first drug or active agent. 6 is attached to the first drug or active agent, if present, or V 6When present, is attached to the first drug or active agent.

[0208] In certain embodiments, the conjugate of Formula (I) comprises a second linker, L B The second linker L B The second moiety of interest (e.g., a second drug or active agent) is attached to the conjugate. The second linker L may be used to link the nucleotide to a polypeptide (e.g., an antibody) via a linking moiety. B may be attached (e.g., covalently attached) to the conjugation moiety (e.g., as described herein). For example, the second linker L B may link a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a second drug or active agent. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be linked to the second linker L B It may be used to conjugate a second drug or active agent (ie, a second agent or active agent) to a polypeptide, such as an antibody.

[0209] For example, as shown in formula (I) above, L B via the conjugation moiety, W 3 may be coupled to W 3 is connected to a second linker, L, via a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety B As mentioned above, W 3 is a polypeptide (e.g., an antibody), i.e., L B may be attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, e.g., a linker L Bmay be indirectly attached to the polypeptide (antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0210] Any convenient linker may be used as the second linker, L, in the subject conjugates and compounds. B In one embodiment, the second linker L B may comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker L B may comprise an alkyl group or a substituted alkyl group. In certain embodiments, the second linker, L B may comprise an alkenyl group or a substituted alkenyl group. In certain embodiments, the second linker, L B may comprise an alkynyl group or a substituted alkynyl group. In certain embodiments, the second linker, L B may comprise an alkoxy group or a substituted alkoxy group. In some embodiments, the second linker, L B may comprise an amino group or a substituted amino group. In certain embodiments, the second linker, L B may contain a carboxyl group or a carboxyl ester group. In one embodiment, the second linker, L B may comprise an acylamino group. In one embodiment, the second linker, L B may comprise an alkylamide group or a substituted alkylamide group. In certain embodiments, the second linker, L B may comprise an aryl group or a substituted aryl group. In certain embodiments, the second linker, L B may comprise a heteroaryl group or a substituted heteroaryl group. In certain embodiments, the second linker, LB may comprise a cycloalkyl group or a substituted cycloalkyl group. In some embodiments, the second linker, L B may include a heterocyclyl group or a substituted heterocyclyl group.

[0211] In one embodiment, the second linker L B The polymer may comprise a polymer. For example, the polymer may comprise polyalkylene glycols and derivatives thereof, such as polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., these homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In some embodiments, the polymer may comprise a polyalkylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., these homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. The polymer is a polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated in the conjugates and compounds described in more detail below.

[0212] In some embodiments, L B is a second linker described by the formula: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m , L 7 , L 8 , L 9 , L 10 , L 11 , L 12 , and L 13are each, independently of one another, a linker subunit; and g, h, i, j, k, l, and m are each, independently of one another, 0 or 1.

[0213] In some embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In some embodiments, the sum of g, h, i, j, k, l, and m is 0. In some embodiments, the sum of g, h, i, j, k, l, and m is 1. In some embodiments, the sum of g, h, i, j, k, l, and m is 2. In some embodiments, the sum of g, h, i, j, k, l, and m is 3. In some embodiments, the sum of g, h, i, j, k, l, and m is 4. In some embodiments, the sum of g, h, i, j, k, l, and m is 5. In some embodiments, the sum of g, h, i, j, k, l, and m is 6. In some embodiments, the sum of g, h, i, j, k, l, and m is 7. In some embodiments, g, h, i, j, k, l, and m are each 1. In some embodiments, g, h, i, j, k, and l are each 1, and m is 0. In some embodiments, g, h, i, j, and k are each 1, and l and m are each 0. In some embodiments, g, h, i, and j are each 1 and k, and l and m are each 0. In some embodiments, g, h, and i are each 1, and j, k, l, and m are each 0. In some embodiments, g and h are each 1, and i, j, k, l, and m are each 0. In some embodiments, g is 1, and h, i, j, k, l, and m are each 0. In some embodiments, g, h, i, j, k, l, and m are each 0.

[0214] In one embodiment, the linker subunit L 7 is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 8 is the second drug or active agent W, if present 2In one embodiment, the linker subunit L 9 is the second drug or active agent W, if present 2 In one embodiment, the linker subunit L 10 is the second drug or active agent W, if present 2 In one embodiment, the linker subunit L 11 is the second drug or active agent W, if present 2 In one embodiment, the linker subunit L 12 is the second drug or active agent W, if present 2 In one embodiment, the linker subunit L 13 is the second drug or active agent W, if present 2 is connected to

[0215] Any convenient linker subunit may be used as the second linker L B Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene, and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclyl groups, combinations thereof, and substituted versions thereof. In some embodiments, L 7 , L 8 , L 9 , L 10 , L 11 , L 12 , and L 13 each (if present) independently comprises one or more groups 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 comprising alkylenediamines).

[0216] In some embodiments, L 7(when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 7 comprises polyethylene glycol. In some embodiments, L 7 comprises a modified polyethylene glycol. In some embodiments, L 7 comprises an amino acid residue. In some embodiments, L 7 In some embodiments, L 7 In some embodiments, L comprises an aryl group or a substituted aryl group. 7 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0217] In some embodiments, L 8 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 8 comprises polyethylene glycol. In some embodiments, L 8 comprises a modified polyethylene glycol. In some embodiments, L 8 comprises an amino acid residue. In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L comprises an aryl group or a substituted aryl group. 8 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0218] In some embodiments, L 9 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 9 comprises polyethylene glycol. In some embodiments, L 9comprises a modified polyethylene glycol. In some embodiments, L 9 comprises an amino acid residue. In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L comprises an aryl group or a substituted aryl group. 9 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0219] In some embodiments, L 10 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 10 comprises polyethylene glycol. In some embodiments, L 10 comprises a modified polyethylene glycol. In some embodiments, L 10 comprises an amino acid residue. In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L comprises an aryl group or a substituted aryl group. 10 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0220] In some embodiments, L 11 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 11 comprises polyethylene glycol. In some embodiments, L 11 comprises a modified polyethylene glycol. In some embodiments, L 11 comprises an amino acid residue. In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L comprises an aryl group or a substituted aryl group.11 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0221] In some embodiments, L 12 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 12 comprises polyethylene glycol. In some embodiments, L 12 contains modified polyethylene glycol In some embodiments, L 12 comprises an amino acid residue. In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L comprises an aryl group or a substituted aryl group. 12 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0222] In some embodiments, L 13 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 13 comprises polyethylene glycol. In some embodiments, L 13 comprises a modified polyethylene glycol. In some embodiments, L 13 comprises an amino acid residue. In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L comprises an aryl group or a substituted aryl group. 13 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0223] In some embodiments, L B is -(L 7 ) g -(L 8 )h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m a second linker comprising -(L 7 ) g -ha-(T 7 -V 7 ) g - and; -(L 8 ) h -ha-(T 8 -V 8 ) h - and; -(L 9 ) i -ha-(T 9 -V 9 ) i - and; -(L 10 ) j -ha-(T 10 -V 10 ) j - and; -(L 11 ) k -ha-(T 11 -V 11 ) k - and; -(L 12 ) l -ha-(T 12 -V 12 ) l - and; -(L 13 ) m -ha-(T 13 -V 13 ) m - and T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 is a linking group, if present; V 7, V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 is a covalent bond or linking functional group, if present; g, h, i, j, k, l, and m are each, independently of one another, 0 or 1.

[0224] In some embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In some embodiments, the sum of g, h, i, j, k, l, and m is 0. In some embodiments, the sum of g, h, i, j, k, l, and m is 1. In some embodiments, the sum of g, h, i, j, k, l, and m is 2. In some embodiments, the sum of g, h, i, j, k, l, and m is 3. In some embodiments, the sum of g, h, i, j, k, l, and m is 4. In some embodiments, the sum of g, h, i, j, k, l, and m is 5. In some embodiments, the sum of g, h, i, j, k, l, and m is 6. In some embodiments, the sum of g, h, i, j, k, l, and m is 7. In some embodiments, g, h, i, j, k, l, and m are each 1. In some embodiments, g, h, i, j, k, and l are each 1, and m is 0. In some embodiments, g, h, i, j, and k are each 1, and l and m are each 0. In some embodiments, g, h, i, and j are each 1, and k, l, and m are each 0. In some embodiments, g, h, and i are each 1, and j, k, l, and m are each 0. In some embodiments, g and h are each 1, and i, j, k, l, and m are each 0. In some embodiments, g is 1, and h, i, j, k, l, and m are each 0. In some embodiments, g, h, i, j, k, l, and m are each 0.

[0225] As noted above, in one embodiment, L 7is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). Thus, in one embodiment, , T 7 is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, V 7 is attached to a second drug or active agent. 8 When present, T is attached to a second drug or active agent. 8 is attached to a second drug or active agent, if present, or V 8 When present, L is attached to a second drug or active agent. 9 When present, T is attached to a second drug or active agent. 9 is attached to a second drug or active agent, if present, or V 9 When present, L is attached to a second drug or active agent. 10 When present, T is attached to a second drug or active agent. 10 is conjugated to a second drug or active agent, if present, or 4 When present, L is attached to a second drug or active agent. 11 When present, T is attached to a second drug or active agent. 11 is attached to a second drug or active agent, if present, or V 11 When present, L is attached to a second drug or active agent. 12 When present, T is attached to a second drug or active agent. 12 is attached to a second drug or active agent, if present, or V12 When present, L is attached to a second drug or active agent. 13 When present, T is attached to a second drug or active agent. 13 is attached to a second drug or active agent, if present, or V 13 When present, is attached to a second drug or active agent.

[0226] T is a linking group 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 With respect to T, any convenient linking group may be utilized in the subject linkers. 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 are each independently a covalent bond, (C1 to C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) xand each w is an integer of 1 to 20, each n is an integer of 1 to 30, each p is an integer of 1 to 20, and each x is an integer of 1 to 12.

[0227] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (C1~C 12 ) alkyl or substituted (C1-C 12 In some embodiments, (C1-C 12 )Alkyl is a straight or branched chain alkyl group containing 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, (C1-C 12 )Alkyl is C1-C 12 Alkyl, or C1-C 10 It may be alkyl or substituted alkyl, such as alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) alkyl is C2 alkyl. For example, (C1-C 12 )Alkyl is C1-C 12 Alkylene or C1-C10 Alkylene, or C1-C6 alkylene, or C1-C3 alkylene In some cases, the alkylene may be an alkylene or a substituted alkylene, such as alkylene (C1-C 12 ) alkyl is C1 alkylene (e.g., CH2). In some cases, (C1-C 12 ) alkyl is C2 alkylene (e.g., CH2CH2). In some cases, (C1-C 12 ) Alkyl is C alkylene (e.g., CH2CH2CH2).

[0228] In some embodiments, the substitution (C1-C 12 ) alkyl is a straight or branched chain substituted alkyl group containing 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, substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkyl or substituted C1-C 10 It may be an alkyl, or a substituted alkyl, such as 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, substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkylene or substituted C1-C 10 It may be alkylene, or substituted alkylene, such as substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some cases, the substituted (C1-C 12 ) alkyl is a substituted C1 alkylene (e.g., a C1 alkylene substituted with —SO3H). In some cases, a substituted (C1-C 12 ) alkyl is a substituted C2 alkylene. In some cases, substituted (C1-C 12 ) alkyl is a substituted C alkylene. For example, substituted (C1-C 12 ) alkyl is (PEG) as described herein.k groups (e.g., -CONH(PEG) such as -CONH(PEG) or -CONH(PEG) k or -NHCO(PEG) such as -NHCO(PEG) k ) C1~C substituted with 12 It may contain alkylene (e.g., C alkylene or C alkylene), or a C1-C substituted with a -CONHCH2CH2SO3H group. 12 It may contain alkylene (e.g., C alkylene) or C-C substituted with -NHCOCH2SO3H groups. 12 It may include alkylene (eg, C5 alkylene).

[0229] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, a linking 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 substituted phenyl. The substituted phenyl is (C1-C 12 ) Alkyl, substituted (C1-C 12) may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some 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 glycoside derivative).

[0230] In some cases, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes heteroaryl or substituted heteroaryl, such as triazolyl (e.g., 1,2,3-triazolyl). In some cases, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes cycloalkyl or substituted cycloalkyl. In some cases, the linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11, T 12 , and / or T 13 ) includes heterocyclyl or substituted heterocyclyl. In some cases, the substituent on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl includes a cleavable moiety as described herein (e.g., an enzyme-cleavable moiety such as a glycoside or glycoside derivative).

[0231] In some embodiments, a linking 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, e.g., an EDA-containing linking group. In some embodiments, (EDA) w comprises one or more EDA moieties, e.g., w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) moieties may be optionally substituted in one or more convenient positions with any convenient substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In one embodiment, the EDA moiety is described by the following structure:

[0232] [ka]

[0233] In the formula, y is an integer of 1 to 6, or 0 or 1, and each R 12are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be linked in a ring, for example, to form a piperazinyl ring. In some embodiments, y is 1 and two adjacent R 12 In one embodiment, y is 1 and adjacent R groups are cyclically linked to form a piperazinyl ring. 12 The groups are selected from hydrogen, alkyl (eg, methyl), and substituted alkyl (eg, lower alkyl-OH such as ethyl-OH or propyl-OH).

[0234] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety can be optionally substituted in one or more convenient positions with any convenient substituent, for example, an 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:

[0235] [ka]

[0236] In the formula, R 12 is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. In one embodiment, R 12 is a carboxy-modified polyethylene glycol.

[0237] In one embodiment, R 12 is represented by the formula (PEG) k which formula may be represented by the following structure:

[0238] [ka] ,

[0239] wherein k is an integer from 1 to 20, e.g., 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some 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, heterocyclyl, and substituted heterocyclyl. 17 is COOH. In one embodiment, R 17 is OH. In some embodiments, R 17 is OCH3.

[0240] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (PEG) n (PEG) n is a polyethylene glycol-linked unit or a modified polyethylene glycol-linked unit. In one embodiment, (PEG) n is described by the following structure:

[0241] [ka] ,

[0242] In the formula, n is an integer of 1 to 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12 , or 1-6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0243] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (AA) p where AA is an amino acid residue. Any convenient amino acid may be utilized. Amino acids of interest include, but are not limited to, L-amino acids and D-amino acids, naturally occurring amino acids such as any of the 20 major α-amino acids and β-alanine, and non-naturally occurring amino acids (e.g., amino acid analogs) such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids. In some embodiments, p is an integer between 1 and 50, e.g., between 1 and 40, 1 and 30, 1 and 20, 1 and 12, or 1 and 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 1. In some embodiments, p is 2.

[0244] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T10 , T 11 , T 12 , and / or T 13 ) include amino acid analogs. Amino acid analogs include compounds similar in structure and / or overall shape to one or more amino acids normally found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as naturally occurring D-forms, as well as L-forms. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, differing in one or more modified groups in the molecule. Such modifications can include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as a methyl group or a hydroxyl group) or atom (such as Cl or Br), deletion of a group, replacement of a covalent bond (such as replacement of a single bond with a double bond), or combinations thereof. For example, amino acid analogs can include alpha-hydroxy acids, alpha-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.

[0245] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is the formula -(CR 13 OH) x-, where x is 0 or x is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 13 is hydrogen. In some embodiments, R 13 is 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~ In one embodiment, R is an alkyl or substituted alkyl, such as a trisubstituted alkyl. 13 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 13 is alkynyl or substituted alkynyl. In some embodiments, R 13 is alkoxy or substituted alkoxy. In some embodiments, R 13 is amino or substituted amino. In one embodiment, R 13 is carboxyl or carboxyl ester. In one embodiment, R 13is acyl or acyloxy. In one embodiment, R 13 is acylamino or aminoacyl. In one embodiment, R 13 is an alkylamide or a substituted alkylamide. In one embodiment, R 13 is sulfonyl. In one embodiment, R 13 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 13 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 13 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 13 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 13 Heterocyclyl or substituted heterocyclyl, for example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0246] In one embodiment, R 13is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are selected from R 13 As mentioned above.

[0247] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the linking group comprises an acetal group. In some embodiments, the linking group comprises a hydrazine. In some embodiments, the linking group comprises a disulfide. In some embodiments, the linking group comprises an ester.

[0248] In some embodiments, a linking group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).

[0249] In some embodiments, the linking group comprises a MABO group, which is described by the following structure:

[0250] [ka]

[0251] In some embodiments, the linking group comprises an MABC group described by the following structure:

[0252] [ka]

[0253] In some embodiments, the linking group comprises a PABO group described by the following structure:

[0254] [ka]

[0255] In some embodiments, the linking group comprises a PABC group described by the following structure:

[0256] [ka]

[0257] In some embodiments, the linking group comprises a PAB group described by the following structure:

[0258] [ka]

[0259] In some embodiments, the linking group comprises a PABA group described by the following structure:

[0260] [ka]

[0261] In some embodiments, the linking group comprises a PAP group described by the following structure:

[0262] [ka]

[0263] In some embodiments, the linking group comprises a PHP group described by the following structure:

[0264] [ka]

[0265] In one embodiment, each R 14 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0266] In one embodiment, R 14 is hydrogen. In one embodiment, each R 14 is hydrogen. In some embodiments, R 14 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 14 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 14 is alkynyl or substituted alkynyl. In some embodiments, R 14 is alkoxy or substituted alkoxy. In some embodiments, R 14 is amino or substituted amino. In one embodiment, R 14 is carboxyl or carboxyl ester. In one embodiment, R 14 is acyl or acyloxy. In one embodiment, R 14 is acylamino or aminoacyl. In one embodiment, R 14 is an alkylamide or a substituted alkylamide. In one embodiment, R 14 is sulfonyl. In one embodiment, R 14 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 14 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 14 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 14 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 14Heterocyclyl or substituted heterocyclyl, for example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0267] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP linked structures shown above, the phenyl 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, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0268] In one embodiment, the linking group T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 Each of one or more of T is optionally substituted with a glycoside or glycoside derivative. For example, in some cases, 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of T is optionally substituted with a glycoside. 7, T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 is optionally substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0269] In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP linked structures shown above 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 linked structures shown above, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0270] For example, in some embodiments, the glycoside or glycoside derivative may be selected from the following structures:

[0271] [ka] ,

[0272] [ka] ,

[0273] [ka] ,

[0274] [ka] ,

[0275] [ka] ,

[0276] [ka] , and

[0277] [ka]

[0278] V, the linking functional group 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 With respect to V, any convenient linking functional group may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, 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 a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where q is an integer from 1 to 6. In some embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6.

[0279] In some embodiments, each R 15 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0280] In one embodiment, R 15 is hydrogen. In one embodiment, each R 15 is hydrogen. In some embodiments, R 15 is 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 1~3In some embodiments, R 15 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 15 is alkynyl or substituted alkynyl. In some embodiments, R 15 is alkoxy or substituted alkoxy. In some embodiments, R 15 is amino or substituted amino. In one embodiment, R 15 is carboxyl or carboxyl ester. In one embodiment, R 15 is acyl or acyloxy. In one embodiment, R 15 is acylamino or aminoacyl. In one embodiment, R 15 is an alkylamide or a substituted alkylamide. In one embodiment, R 15 is sulfonyl. In one embodiment, R 15 is thioalkoxy or substituted thioalkoxy. In some embodiments, R 15 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 15 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 15 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 15 Heterocyclyl or substituted heterocyclyl, for example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0281] In one embodiment, each R 15 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted Alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are defined as R 15 As mentioned above.

[0282] As noted above, in some embodiments, L A is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 )e -(T 6 -V 6 ) f -, and a, b, c, d, e, and f are each, independently of one another, 0 or 1.

[0283] In some embodiments, the first linker L A In T 1 is (C1~C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 are each independently of each other, (C1~C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 are each independently a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15SO2-, and -P(O)OH-, where q is an integer from 1 to 6;

[0284] (PEG) n teeth

[0285] [ka] where n is an integer from 1 to 30;

[0286] EDA is an ethylenediamine moiety having the following structure:

[0287] [ka] , y is an integer from 1 to 6, and r is 0 or 1;

[0288] 4-amino-piperidine (4AP)

[0289] [ka] and;

[0290] AA is an amino acid residue, and p is an integer from 1 to 20; Each R 12 are each independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring; Each R 13 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0291] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is selected from the following: T 1 (C1~C 12 ) alkyl, and V 1 is -CO-; T 2 is an amino acid analogue, and V 2 is -NH-; T 3 (PEG) n and V 3 is -CO-; T 4 is AA and V 4 does not exist; T 5 is PABC and V 5 does not exist; f is 0; or T 1 (C1~C 12 ) alkyl, and V 1 is -CONH-; T 2 (PEG) n and V 2 is -CO-; T 3 is AA and V 3 does not exist; T 4 is PABC and V 4does not exist; e and f are each 0; or T 1 (C1~C 12 ) alkyl, and V 1 is -CONH-; T 2 is a substitution (C1~C 12 ) alkyl, and V 2 is -CO-; T 3 is AA and V 3 does not exist; T 4 is PABC and V 4 does not exist; e and f are each 0.

[0292] In one embodiment, the first linker L A the left-hand side of the linker structure is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and a first linker L A The right-hand side of the linker structure is attached to a first drug or active agent.

[0293] As noted above, in some embodiments, L B is -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) m -, and g, h, i, j, k, l, and m are each, independently of one another, 0 or 1.

[0294] In some embodiments, the second linker L B In: T 7 is (C1~C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each independently of each other, (C1~C 12 ) Alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 are each independently a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2-, and -P(O)OH-, where q is an integer from 1 to 6;

[0295] (PEG) n teeth,

[0296] [ka] where n is an integer from 1 to 30;

[0297] EDA is an ethylenediamine moiety having the following structure:

[0298] [ka] , y is an integer from 1 to 6, and r is 0 or 1;

[0299] 4-amino-piperidine (4AP)

[0300] [ka] and;

[0301] AA is an amino acid residue, and p is an integer from 1 to 20; Each R 12 are each independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring; Each R 13 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 are each independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkoxy, cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0302] Any convenient linking group may be T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 For example, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Any of the linking groups described above with respect to the linking group T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 It may be used for

[0303] Any convenient linking functional group may be V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 For example, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 Any of the linking functional groups described above with respect to the linking functional group V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 It may be used for

[0304] In one embodiment, each R 13 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are selected from R 13 As mentioned above.

[0305] In one embodiment, each R 15 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are each independently selected from R 15 In these embodiments, the various possible substituents are as described above for R 15 As mentioned above.

[0306] Second linker L B In some embodiments, the linking group T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 wherein one or more of each is optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0307] Second linker L BIn certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP linked structures shown above 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 linked structures shown above, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0308] In one embodiment, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 , and V 7 , V 8 , V 9 , V 10 ,V 11 , V 12 , and V 13 is selected from the following: T 7 does not exist, and V 7 is -NHCO-; T 8 (C1~C 12 ) alkyl, and V 8 is -CONH-; T 9 (PEG) n and V 9 is -CO-; T 10 is AA and V 10 does not exist; T 11 is PABC and V 11 does not exist; l and m are each 0; or T 7 does not exist, and V 7is -NHCO-; T 8 (C1~C 12 ) alkyl, and V 8 is -CONH-; T 9 is a substitution (C1~C 12 ) alkyl, and V 9 is -CO-; T 10 is AA and V 10 does not exist; T 11 is PABC and V 11 does not exist; l and m are each 0.

[0309] In one embodiment, the second linker L B the left hand side of the above linker structure is attached to a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and a second linker L B The right hand side of the linker structure is attached to a second drug or active agent.

[0310] In certain embodiments, the conjugate is an antibody-drug conjugate in which the antibody and one or more drugs or active agents are linked by a linker, as described above. In some cases, the linker (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, which include one or more bonds that can dissociate under certain conditions to separate the cleavable linker into two or more separable parts. For example, a cleavable moiety can include one or more covalent bonds that can dissociate or degrade under certain conditions to separate the cleavable linker into two or more parts. Thus, a linker included in an antibody-drug conjugate can be a cleavable linker that can be cleaved under appropriate conditions to separate or release a drug from an antibody at a desired target site of action for the drug.

[0311] In some cases, a cleavable linker comprises two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties may be configured such that cleavage of both cleavable moieties is necessary to separate or release the drug from the antibody at the desired target site of action for the drug. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that impedes cleavage of the first cleavable moiety. By "impedes cleavage," we mean that the presence of an uncleaved second cleavable moiety reduces or significantly inhibits cleavage of the first cleavable moiety, thereby significantly reducing or preventing cleavage of the cleavable linker. For example, the presence of an uncleaved second cleavable moiety may impede cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety is prevented by the presence of the second cleavable moiety, which significantly reduces or prevents release of the drug from the antibody, e.g., premature release of the drug from the antibody can be significantly reduced or prevented until the antibody-drug conjugate reaches or near the desired target site of action for the drug.

[0312] In some cases, because the second cleavable moiety impedes cleavage of the first cleavable moiety, cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety, followed by cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the impediment to cleavage of the first cleavable moiety, thereby rendering the first cleavable moiety cleavable. Cleavage of the first cleavable moiety can result in dissociation or separation of the cleavable linker into two or more moieties, as described above, and release of the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially, we mean that in the presence of an uncleaved second cleavable moiety, It means that about 10% or less first cleavable moiety cleavage occurs, such as 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 first cleavable moiety cleavage occurs in the presence of an uncleaved second cleavable moiety.

[0313] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, thereby significantly reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate reaches or near the drug's desired target site of action. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), rendering the first cleavable moiety cleavable, thereby cleaving the cleavable linker and separating or releasing the drug from the antibody at the drug's desired target site of action, as described above. In certain cases, although cleavage of the second cleavable moiety exposes the first cleavable moiety for subsequent cleavage, cleavage of the second cleavable moiety does not itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is further required to cleave the cleavable linker).

[0314] Each cleavable moiety included in the cleavable linker may be an enzyme-cleavable moiety. For example, the first cleavable moiety may be a first enzyme-cleavable moiety, and the second cleavable moiety may be a second enzyme-cleavable moiety. The enzyme-cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above through the enzymatic action of an enzyme. The enzyme-cleavable moiety may be any cleavable moiety that can be cleaved through the enzymatic action of an enzyme, such as, but not limited to, an ester, a peptide, or a glycoside. In some cases, the enzyme that cleaves the enzyme-cleavable moiety is present at the desired target site of action, e.g., the desired target site of action of the drug to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzyme-cleavable moiety is present in lesser amounts in other regions, such as whole blood, plasma, or serum. In this way, cleavage of the enzyme-cleavable moiety can be controlled so that significant cleavage occurs at the desired site of action, but little cleavage occurs in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0315] For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, such as to deliver a cancer therapeutic agent to a desired site of action in cancer cells. In some cases, enzymes such as esterases, which cleave ester bonds, or glycosidases, which cleave glycosidic bonds, can be overexpressed in cancer cells and serve as biomarkers for cancer. The overexpression, i.e., localization, of certain enzymes in cancer can be utilized in conjunction with an enzyme-cleavable moiety included in the cleavable linker of the antibody-drug conjugates of the present disclosure to specifically release a drug at a desired site of action (i.e., the location of the cancer (and the overexpressed enzyme)). That is, in some embodiments, the enzyme-cleavable moiety is a cleavable moiety (e.g., an ester or glycoside) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be an esterase. Thus, in some cases, the enzyme-cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme. In some cases, the enzyme can be a glycosidase. Thus, in some cases, the enzyme-cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme.

[0316] In one embodiment, the enzyme-cleavable moiety is an ester bond. For example, the first cleavable moiety (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) is , an ester. The presence of an uncleaved second cleavable moiety protects the first cleavable moiety (ester) from cleavage by esterase enzymes, thereby significantly reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate reaches or near the desired target site of action for the drug. In some cases, a portion of the linker adjacent to the first cleavable moiety is linked to or includes a substituent that includes a second cleavable moiety. In some cases, the second cleavable moiety includes a glycoside or glycoside derivative.

[0317] In some embodiments, the enzyme-cleavable moiety is a sugar moiety, such as a glycoside (or glyosyl) or a glycoside derivative. In some cases, the glycoside or glycoside derivative may facilitate increased hydrophilicity of the cleavable linker when compared to a cleavable linker that does not contain a glycoside or glycoside derivative. The glycoside or glycoside derivative may be any glycoside or glycoside derivative that is suitable for use in a cleavable linker and can be cleaved via 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) may be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable moiety comprises an ester and the second cleavable moiety comprises a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable moiety is a glucuronide. In some cases, the second cleavable moiety is a galactoside. In some cases, the second cleavable moiety is a glucoside. In some cases, the second cleavable moiety is a mannoside. In some cases, the second cleavable moiety is a fucoside. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0318] A glycoside or glycoside derivative may be covalently bonded to a cleavable linker via a glycosidic bond. The glycosidic bond may be linked 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 may be enzymatically cleaved from the cleavable linker to which it is attached (e.g., through enzyme-mediated hydrolysis of the glycosidic bond). The glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme capable of cleaving (hydrolyzing) the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. An example of an enzyme that can be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker is a glycosidase, such as glucuronidase, galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes may also be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker is one that is located at or near the desired site of action for the drug in the antibody-drug conjugate. For example, the enzyme may be a lysosomal enzyme, such as an intralysosomal glycosidase, that is present in cells at or near the desired site of action for the drug of the antibody drug conjugate. In some cases, the enzyme may be a lysosomal glycosidase that is present in cells at or near the desired site of action for the drug of the antibody drug conjugate. The enzyme mediating cleavage is present at or near the target site.

[0319] Examples of conjugates according to the present disclosure include, but are not limited to, the following structures:

[0320] [ka] ,

[0321] [ka] , and

[0322] [ka]

[0323] Any of the chemicals, linkers and conjugation moieties described in the structures above can be adapted for use in the subject compounds and conjugates.

[0324] Further disclosure regarding hydrazinyl-indolyl compounds and hydrazinyl-pyrrolo-pyridinyl compounds, as well as methods for preparing conjugates, can be found in U.S. Patent Nos. 9,310,374 and 9,493,413, the disclosures of each of which are incorporated herein by reference. Further disclosure regarding cleavable linkers can be found in International Publication Nos. WO 2020 / 154437, filed January 22, 2020, PCT / US2021 / 060193, filed November 19, 2021, and PCT / US2022 / 012347, filed January 13, 2022, the disclosures of which are incorporated herein by reference. Further disclosure regarding branched linkers can be found in International Publication No. WO 2020 / 154437, filed January 22, 2020, PCT / US2021 / 060193, filed November 19, 2021, and PCT / US2022 / 018534, filed March 2, 2022, the disclosures of which are incorporated herein by reference.

[0325] Compounds useful for preparing conjugates The present disclosure provides compounds useful for producing the conjugates described herein. In certain embodiments, the compounds may be conjugated to one or more drugs or active agents, and may include a hydrazinyl-indolyl conjugation moiety or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety useful for conjugating one or more drugs or active agents to a polypeptide (e.g., an antibody). For example, the conjugation moiety in the compounds may be conjugated to a polypeptide (e.g., an antibody) to indirectly link one or more drugs or active agents to the polypeptide (antibody).

[0326] In one embodiment, the compound is a compound of formula (II):

[0327] [ka] (II)

[0328] During the ceremony, Z 1 , Z 2 , Z 3 , and Z 4 are each, independently of each other, 4 , N, and C.L. B -W 2 Selected from; R 2 and R 3 are each, independently of one another, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3are optionally joined in a ring to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker; L B is a second linker; W 1 is the first drug; W 2 is the second drug.

[0329] With respect to compounds of formula (II), the substituent Z 1 , Z 2 , Z 3 , Z 4 , R 2 , R 3 , R 4 , L A , L B , W 1 , and W 2 is as described above for the conjugate of formula (I). Similarly, the first linker L of formula (II) A and a second linker L B Regarding T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 , and T 7 , T 8 , T 9 , T10 , T 11 , T 12 , T 13 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 The substituents are as described above for the conjugate of formula (I).

[0330] For example, in some cases, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is selected from: T 1 (C1~C 12 ) alkyl, and V 1 is -CO-; T 2 is an amino acid analogue, and V 2 is -NH-; T 3 (PEG) n and V 3 is -CO-; T 4 is AA and V 4 does not exist; T 5 is PABC and V 5 does not exist; f is 0; or T 1 (C1~C 12 ) alkyl, and V 1 is -CONH-; T 2 (PEG) n 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; e and f are each 0; or T 1 (C1~C 12 ) alkyl, and V 1 is -CONH-; T 2 is a substitution (C1~C 12 ) alkyl, and V 2 is -CO-; T 3 is AA and V 3 does not exist; T 4 is PABC and V 4 does not exist; e and f are each 0.

[0331] For example, in some cases, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 , and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 is selected from: T 7 does not exist, and V 7 is -NHCO-; T 8 (C1~C 12 ) alkyl, and V 8 is -CONH-; T 9 (PEG) n and V 9 is -CO-; T 10 is AA and V 10 does not exist; T 11 is PABC and V 11 does not exist; l and m are each 0; or T 7 does not exist, and V 7 is -NHCO-; T 8 (C1~C 12 ) alkyl, and V 8 is -CONH-; T 9 is a substitution (C1~C 12 ) alkyl, and V 9 is -CO-; T 10 is AA and V 10 does not exist; T 11 is PABC and V 11 does not exist; l and m are each 0.

[0332] Compounds of formula (II) may be used in the conjugation reactions described herein to conjugate one or more drugs or active agents attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a polypeptide (e.g., an antibody) to form an antibody-drug conjugate.

[0333] Examples of compounds according to the present disclosure include, but are not limited to, the following structures:

[0334] [ka] ,

[0335] [ka] , and

[0336] [ka]

[0337] Any of the chemicals, linkers and conjugation moieties described in the structures above can be adapted for use in the subject compounds and conjugates.

[0338] Anti-nectin-4 antibody As noted above, the subject complexes may be formed by the addition of substituents W 2 The amino acid sequence of the anti-Nectin-4 antibody may be modified to include a 2-formylglycine (fGly) residue. 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. They may be referred to by abbreviations such as: alanine or Ala or A; cysteine ​​or Cys or C; aspartic acid or Asp or D; glutamic acid or Glu or E; phenylalanine or Phe or F; glycine or Gly or G; histidine or His or H; isoleucine or Ile or I; lysine or Lys or K; leucine or Leu or L; methionine or Met or M; asparagine or Asn or N; proline or Pro or P; glutamine or Gln or Q; arginine or Arg or R; serine or Ser or S; threonine or Thr or T; valine or Val or V; tryptophan or Trp or W; and tyrosine or Tyr or Y.

[0339] The various protein sequences identified in this disclosure are provided below in Table 2 and are referenced herein based on their sequence identifiers (SEQ ID NOs).

[0340] Table 2. Heavy chain variable region (VH) and light chain variable region (VL) sequences and sequence identifiers for various antibodies disclosed herein. The first, second, and third CDR sequences in each VH and VL sequence are bolded and underlined. The first framework (FW1) region precedes the first CDR, the second framework (FW2) region follows the first CDR, the third framework (FW3) region follows the second CDR, and the fourth framework (FW4) region follows the third CDR.

[0341] [Table 2-1]

[0342] Table 2 continued [Table 2-2]

[0343] Table 2 continued [Table 2-3]

[0344] Table 2 continued [Table 2-4]

[0345] The CDR sequences and corresponding sequence identifiers are shown in Table 3 below:

[0346] [Table 3-1]

[0347] Table 3 continued [Table 3-2]

[0348] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0349] A heavy chain variable region (VH) chain comprising heavy chain CDRs 1 to 3 (HCDRs 1 to 3) of a VH chain having a sequence selected from SEQ ID NOs: 1 to 17; and

[0350] A light chain variable region (VL) chain comprising VL chain light chain CDR1 to 3 (LCDR1 to 3) having a sequence selected from SEQ ID NOs: 18 to 31.

[0351] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0352] a VH chain comprising a sequence selected from SEQ ID NOs: 1 to 17; and

[0353] A VL chain comprising a sequence selected from SEQ ID NOs: 18 to 31.

[0354] In some cases, the antibodies of the present disclosure specifically bind to Nectin-4, including:

[0355] A VH chain comprising HCDRs 1 to 3 of a VH chain having a sequence selected from SEQ ID NOs: 1 to 6; and

[0356] A VL chain comprising LCDR1 to LCDR3 of a VL chain having a sequence selected from SEQ ID NOs: 18 to 23.

[0357] That is, in certain embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0358] A VH chain comprising a sequence selected from SEQ ID NOs: 1 to 6; and

[0359] A VL chain comprising a sequence selected from SEQ ID NOs: 18 to 23.

[0360] Antibodies comprising any combination of heavy and light chains shown in Table 4 below are contemplated.

[0361] [Table 4]

[0362] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0363] A VH chain comprising HCDRs 1 to 3 of a VH chain having a sequence selected from SEQ ID NOs: 7 to 13; and

[0364] A VL chain comprising LCDR1 to LCDR3 of a VL chain having a sequence selected from SEQ ID NOs: 24 to 27.

[0365] In some cases, the antibodies of the present disclosure specifically bind to Nectin-4, including:

[0366] A VH chain comprising a sequence selected from SEQ ID NOs: 7 to 13; and

[0367] A VL chain comprising a sequence selected from SEQ ID NOs: 24 to 27.

[0368] In certain embodiments, antibodies comprising any combination of heavy and light chains shown in Table 5 below are contemplated.

[0369] [Table 5]

[0370] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0371] A VH chain comprising HCDRs 1 to 3 of a VH chain having the sequence of SEQ ID NO: 14; and

[0372] A VL chain comprising LCDR1 to LCDR3 of a VL chain having the sequence of SEQ ID NO:28.

[0373] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and: include:

[0374] A VH chain comprising the sequence of SEQ ID NO: 14; and

[0375] A VL chain comprising the sequence of SEQ ID NO: 28.

[0376] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0377] A VH chain comprising HCDRs 1 to 3 of a VH chain having the sequence of SEQ ID NO: 15; and

[0378] A VL chain comprising LCDR1 to LCDR3 of a VL chain having the sequence of SEQ ID NO:29.

[0379] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0380] A VH chain comprising the sequence of SEQ ID NO: 15; and

[0381] A VL chain comprising the sequence of SEQ ID NO: 29.

[0382] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0383] A VH chain comprising HCDRs 1 to 3 of a VH chain having the sequence of SEQ ID NO: 16; and

[0384] A VL chain comprising LCDR1 to LCDR3 of a VL chain having the sequence of SEQ ID NO:30.

[0385] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0386] A VH chain comprising the sequence of SEQ ID NO: 16; and

[0387] A VL chain comprising the sequence of SEQ ID NO: 30.

[0388] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0389] A VH chain comprising HCDRs 1 to 3 of a VH chain having the sequence of SEQ ID NO: 17; and

[0390] A VL chain comprising LCDR1 to LCDR3 of a VL chain having the sequence of SEQ ID NO:31.

[0391] According to some embodiments, the antibodies of the present disclosure specifically bind to Nectin-4 and include:

[0392] A VH chain comprising the sequence of SEQ ID NO: 17; and

[0393] A VL chain comprising the sequence of SEQ ID NO: 31.

[0394] In one embodiment, the VH chain of the anti-Nectin-4 antibody comprises HCDRs 1 to 3 of a VH chain having a sequence selected from SEQ ID NOs: 1 to 17, and The VH chain of an anti-Nectin-4 antibody of the present disclosure includes an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-17. In certain embodiments, any amino acid differences between the VH chain of an anti-Nectin-4 antibody of the present disclosure and a sequence selected from SEQ ID NOs: 1-17 may be limited to regions outside the CDRs, for example, one or more of the framework regions (FRs), such as FR1, FR2, FR3, and / or FR4.

[0395] In one embodiment, the VL chain of the anti-Nectin-4 antibody comprises LCDR1 to LCDR3 of a VL chain having a sequence selected from SEQ ID NOs: 18 to 31, and comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 18 to 31.

[0396] In certain embodiments, any amino acid differences between the VL chain of an anti-Nectin-4 antibody of the present disclosure and a sequence selected from SEQ ID NOs: 18 to 31 may be limited to regions outside the CDRs, for example, one or more of FR1, FR2, FR3, and / or FR4.

[0397] In one embodiment, the anti-Nectin-4 antibody of the present disclosure comprises: a) a heavy chain comprising a VH region having an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 1 to 17, and a heavy chain constant region having an amino acid sequence set forth in any one of SEQ ID NOs: 70 to 86, in which C present in the sequence LCTPSR in the constant region is replaced with fGly; and b) a light chain comprising a VL region having an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 18 to 31.

[0398] In certain embodiments, the anti-Nectin-4 antibody of the present disclosure comprises: a) a VH region comprising an amino acid sequence having at least 85% identity (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 1 to 17; and a VH region comprising an amino acid sequence having at least 85% identity (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 86. and a) a heavy chain comprising a heavy chain constant region comprising an amino acid sequence having the formula:

[0399] The anti-Nectin-4 antibody of the present disclosure can bind to Nectin-1 protein, e.g., recombinant Nectin-4 protein, with an EC50 of about 0.1-1 nM, e.g., 0.2-0.9 nM, 0.3-0.7 nM, or 0.4-0.6 nM, as measured by ELISA. The concentration of antibody that provides a half maximal response (e.g., half of the maximal fluorescence intensity) is measured as EC50.

[0400] The anti-Nectin-4 antibody of the present disclosure may bind to cancerous tissue and may exhibit no binding or only slight binding to normal tissue (e.g., only slight binding as measured by immunohistochemistry or undetectable binding by immunohistochemistry). For example, the anti-Nectin-4 antibody described herein may bind to human solid tumors, such as ovarian cancer, breast ductal carcinoma, lung adenocarcinoma, and pancreatic cancer, which contain cancerous cells, while exhibiting no detectable binding to human normal tissues, such as ovary, breast, lung, and pancreas, which do not contain cancerous cells.

[0401] These antibodies are useful in a variety of research, diagnostic, and therapeutic applications, including for performing any of the methods described in U.S. Patent Application Publication Nos. 20210130459, 20200231670, 20180243434, 20110301056, 20100285597, and 20080268476, the disclosures of each of which are incorporated herein by reference in their entireties.

[0402] The subject antibodies specifically bind to Nectin-4 polypeptide, the epitope of which includes amino acid residues within the human Nectin-4 antigen, which comprises the amino acid sequence set forth in SEQ ID NO:99:

[0403] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRV STFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLW HIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 99).

[0404] In certain embodiments, the Nectin-4 epitope bound by the anti-Nectin-4 antibodies disclosed herein is present on Nectin-4 expressed by HEK cells overexpressing human Nectin-4 or SK-BR-3 breast cancer cells.

[0405] The subject antibodies exhibit high affinity binding to Nectin-4. For example, the subject antibodies exhibit high affinity binding to Nectin-4, with an affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M, or 10 -12 The subject antibodies bind to epitopes present on Nectin-4 with an affinity of greater than about 10 -7 M ~ about 10 -8 M, about 10 -8M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, or about 10 -11 M~about 10 -12 M, or 10 -12 It binds with an affinity greater than M.

[0406] The anti-Nectin-4 antibodies of the present disclosure may, in some cases, induce apoptosis in cells expressing Nectin-4 on the cell surface.

[0407] A "Nectin-4 antigen" or "Nectin-4 polypeptide" may 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:99.

[0408] As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include the kappa constant region, lambda constant region, alpha constant region (IgA1 and IgA2), gamma constant region (IgG1, IgG2, IgG3, IgG4 ), delta constant region, epsilon constant region, and mu constant region genes; as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains (approximately 25 kD or 214 amino acids) are encoded by a variable region gene (approximately 110 amino acids) at the N-terminus and a kappa or lambda constant region at the C-terminus. Full-length immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) are encoded by a variable region gene (approximately 116 amino acids) at the N-terminus and one of the other constant region genes listed above, e.g., the gamma constant region gene (encoding approximately 330 amino acids), at the C-terminus. In some embodiments, the subject antibodies comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0409] In some embodiments, the subject antibodies do not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but instead comprise an antigen-binding fragment of a full-length immunoglobulin heavy chain and an antigen-binding fragment of a full-length immunoglobulin light chain. In some embodiments, these antigen-binding fragments are comprised on separate polypeptide chains; in other embodiments, these antigen-binding fragments are comprised within a single polypeptide chain. The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody capable of specifically binding to Nectin-4, as described above. Examples of binding fragments include: (i) a Fab fragment (a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region). (iii) Fd fragments (consisting of the VH and CH1 domains); (iv) Fv fragments (consisting of the VH and VL domains of one arm of an antibody); (v) dAb fragments (consisting of the VH domain); (vi) isolated CDRs; (vii) single-chain Fvs (scFvs) (consisting of the VH and VL domains of one arm of an antibody joined by a synthetic linker using recombinant means, such that the pair of VH and VL domains forms a monovalent molecule); (viii) diabodies (consisting of two scFvs in which the VH and VL domains are joined in such a way that they do not pair together to form a monovalent molecule; the VH of one scFv and the VL domain of the other scFv form a bivalent molecule); and (ix) diabodies (consisting of at least two antigen-binding regions, each region binding to a different epitope). In some embodiments, a subject antibody fragment is a Fab fragment. In some embodiments, a subject antibody fragment is a single-chain antibody (scFv).

[0410] In some embodiments, the subject antibodies are recombinant or engineered antibodies, e.g., chimeric, humanized, deimmunized, or in vitro-generated antibodies. As used herein, the terms "recombinant" or "engineered" antibodies are intended to encompass all antibodies produced, expressed, engineered, 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 combinatorial library of recombinant antibodies; (iii) antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes; or (iv) antibodies produced, expressed, engineered, or isolated by any other means, including splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies include humanized antibodies, CDR-grafted antibodies, chimeric antibodies, deimmunized antibodies, and in vitro-generated antibodies; and may optionally include constant regions derived from human germline immunoglobulin sequences.

[0411] Full-length bispecific antibodies can be generated, for example, in a cell-free environment in vitro or by co-expression, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface within each half molecule to promote heterodimerization of two antibody half molecules with different specificities. This Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. A free cysteine ​​from one of the resulting parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue in a second parent monospecific antibody molecule, while the CH3 domains of the parent antibody are released and reassembled by dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a different epitope.

[0412] The "knob-in-hole" strategy (see, e.g., PCT Publication WO 2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domain in human IgG may be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of these two antibodies, heterodimers are formed as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob." Exemplary CH3 substitution pairs that form a knob and hole are (expressed as altered position in the first CH3 domain of the first heavy chain / altered position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S / L368A / Y407V.

[0413] Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface with negatively charged residues on the second CH3 surface, as described in U.S. Patent Application Publication Nos. 2010 / 0015133; 2009 / 0182127; 2010 / 028637, or 2011 / 0123532. In another strategy, heterodimerization may be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain), as described in U.S. Patent Application Publication Nos. 2012 / 0149876 or 2013 / 0195849: L351Y / F405A / Y407V / T394W, T366I / K. 392M / 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.

[0414] Also provided are single-chain bispecific antibodies. 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 completely derived from human immunoglobulins.

[0415] Methods for producing humanized antibodies are known in the art. For example, mouse CDRs can be substituted into a human variable domain framework, so that the human variable domain framework retains the correct spatial orientation, adopting the same or a similar conformation as the mouse variable framework from which the CDRs were derived. This can be achieved by obtaining human variable domains from human antibodies whose framework sequences share a high degree of sequence identity with the mouse variable framework domains from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. These human antibody sequences can be those of naturally occurring human antibodies or consensus sequences of several human antibodies.

[0416] Once the complementarity-determining regions of the mouse donor immunoglobulin and the appropriate human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues should be substituted from these components to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with mouse amino acid residues should be minimized, as the introduction of mouse residues increases the risk that the antibody will elicit a human anti-mouse antibody (HAMA) response in humans. HAMA responses can be monitored in specific patients or during clinical trials using art-recognized methods for measuring immune responses. Patients receiving a humanized antibody can be evaluated for immunogenicity at the start of treatment and throughout its administration. HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples obtained from the patient using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0417] Certain amino acids are selected for substitution from human variable region framework residues based on their potential impact on CDR conformation and / or antigen binding. Unnatural proximity between the mouse CDR region and the human variable framework region can result in unnatural conformational constraints, which, unless corrected by substitution of certain amino acid residues, will result in loss of binding affinity. The selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Typically, molecular models are created starting from the elucidated structure of an immunoglobulin chain or its domain. The amino acid sequence similarity of the chain to be modeled is compared with that of the chain or domain of the elucidated three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for constructing the molecular model. Chains or domains that share at least 50% sequence identity are selected for modeling, and preferably those that share at least 60%, at least 70%, at least 80%, at least 90%, or more. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modeled and those in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and further by ensuring that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0418] Amino acids present in the murine antibody may be selected for substitution into the humanized antibody, provided that framework residues, as defined by Kabat, supra, constitute structural loop residues, as defined by Chothia, supra. Residues "adjacent to a CDR region" include residues located immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., CDRs as defined by Kabat, or Chothia (e.g., Chothia and Lesk JMB 196:901). These include amino acid residues at positions immediately adjacent to the CDRs as defined by the IFN-γ standard (see Amit et al., 1987). These amino acids are particularly likely to interact with amino acids within the CDRs and, if selected from the acceptor, are likely to distort the donor CDRs and reduce affinity. Additionally, these adjacent amino acids may directly interact with the antigen (see Amit et al., 1987). al., Science, 233:747 (1986)), the selection of these amino acids from the donor may be desirable to preserve all of the antigen contacts that confer affinity to the original antibody.

[0419] In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibodies are scFv dimers (e.g., comprising two tandem scFvs (scFv2)), scFv trimers (e.g., comprising three tandem scFvs (scFv3)), scFv tetramers (e.g., comprising four tandem scFvs (scFv4)), or multimers of more than four scFvs (e.g., tandem). The scFv monomers can be linked in tandem via a linker of about 2 to about 10 amino acids in length, e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkers include, for example, (Gly) where x is an integer between 2 and 10. x , glycine-serine polymers, etc.

[0420] In some embodiments, a subject antibody comprises an immunoglobulin constant region (e.g., an Fc region). The Fc region, if present, may be a human Fc region. If a constant region is present, the antibody may contain both a light chain constant region and a heavy chain constant region. The antibodies described herein include antibodies with all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, as well as antibodies with any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is an Fc of human isotype IgG1. The light chain constant region may be λ or κ. A subject antibody (e.g., a subject humanized antibody) may comprise sequences from more than one class or isotype. An antibody may be expressed as a tetramer containing two light chains and two heavy chains; as separate heavy chains, separate light chains; as Fab, Fab', F(ab'), and Fv; or as a single-chain antibody in which the heavy and light chain variable domains are linked via a spacer.

[0421] In some embodiments, anti-Nectin-4 antibodies of the present disclosure may comprise one or more amino acid substitutions introduced into the Fc region. In some embodiments, one or more of the amino acid substitutions may be present at positions 239, 298, 326, 330, and 332 within the Fc region. In some embodiments, anti-Nectin-4 antibodies of the present disclosure may comprise 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.

[0422] In some embodiments, the subject antibodies comprise a free thiol (—SH) group at the carboxyl terminus, in which case the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibodies), a scaffold, a carrier, etc.

[0423] In some embodiments, the subject antibodies comprise one or more unnatural amino acids. In some embodiments, the non-naturally encoded amino acids comprise a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. The inclusion of the unnatural amino acids allows for linkage to a polymer, a second polypeptide, a scaffold, or the like. Examples of such unnatural amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0424] The present disclosure also provides anti-Nectin-4 antibodies conjugated with a moiety of interest, such as a detectable label, a drug, a half-life extending group, etc. Antibody modification can be achieved by a variety of synthetic and / or recombinant methods. The one or more moieties attached to the antibody can provide one or more of a wide variety of functions or characteristics. Exemplary moieties include: detectable labels (e.g., dye labels (e.g., chromophores, fluorophores, etc.)); fluorophores), biophysical probes (spin labels, nuclear magnetic resonance (NMR) probes), fluorescence resonance energy transfer (FRET) labels (e.g., at least one member of a FRET pair (comprising at least one member of a fluorophore / quencher pair)), bioluminescence resonance energy transfer (BRET) labels (e.g., at least one member of a BRET pair), immunodetectable tags (e.g., FLAG, His(6), etc.); water-soluble polymers (e.g., PEGylation); purification tags (e.g., to facilitate isolation by affinity chromatography (e.g., attachment of a FLAG epitope); membrane localization domains (e.g., lipid or glycophosphatidylinositol (GPI)-type anchors); immobilization tags (e.g., to facilitate binding (including selective binding) of a polypeptide to a surface); drugs (e.g., to facilitate drug targeting, such as via conjugation of a drug to an antibody); and the like.

[0425] In some embodiments, the subject antibodies are linked (e.g., covalently linked) 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 natural polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene polymers, polyalkenylene polymers, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.Suitable polymers include, for example, ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoesters; polyanhydrides; polyglycolic acid; poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphate esters. esters; polyphosphate urethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonates); copoly(ether-esters) (e.g., poly(ethylene oxide)-polylactic acid (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene-alpha-olefin copolymers; α-olefins. Examples of suitable polymers include acrylic polymers and copolymers; polymers and copolymers of vinyl halides such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyvinyl aromatics such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers and olefins such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; polyamides such as nylon 66 and polycaprolactam; alkyd resins; polycarbonate; polyoxymethylene; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; propionyl cellulose; cellulose ethers; amorphous teflon; poly(ethylene glycol); and carboxymethyl cellulose.

[0426] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and derivatives thereof, such as substituted poly(ethylene glycol), e.g., methoxypoly(ethylene glycol), and derivatives thereof. Suitable natural polymers include, e.g., albumin, amylose, dextran, glycogen, and derivatives thereof. Derivatives thereof include:

[0427] Suitable polymers can have an average molecular weight within the range of 500 Da to 50,000 Da, e.g., 5,000 Da to 40,000 Da, or 25,000 to 40,000 Da. For example, in some embodiments, when a subject antibody comprises a poly(ethylene glycol) (PEG) polymer or a methoxypoly(ethylene glycol) polymer, the PEG polymer or methoxypoly(ethylene glycol) polymer can have a molecular weight within 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.

[0428] In some embodiments, a subject antibody is covalently linked to a PEG polymer. In some embodiments, a subject scFv multimer is covalently linked to a PEG polymer. PEG suitable for conjugation to proteins is generally water-soluble at room temperature and has the general formula R(O-CH-CH). n The PEG conjugated to the subject antibody may be a linear or branched PEG. Branched PEG derivatives include star PEG and multi-arm PEG.

[0429] The subject antibodies may be glycosylated, e.g., the subject antibodies may comprise covalently linked carbohydrate or polysaccharide moieties. Glycosylation of antibodies is typically either N-linked or O-linked. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so that it contains an N-linked or O-linked glycosylation site. Similarly, removal of glycosylation sites can be accomplished by altering amino acids within the antibody's native glycosylation sites.

[0430] The subject antibodies can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the subject antibodies, a synthetic polymer, a carbohydrate chain, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides via their amino groups. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive groups and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and homobifunctional imidoesters crosslink amine-containing polypeptides. At slightly alkaline pH, imidoesters react only with primary amines to form imidoamides, leaving the overall charge of the crosslinked polypeptides unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propinoamidobutane (DPDPB).

[0431] Heterobifunctional crosslinkers have two or more different reactive groups (e.g., an amine-reactive group and a sulfhydryl-reactive group) and crosslink one polypeptide via the amine-reactive group or the sulfhydryl-reactive group, and then react with the other polypeptide via the unreacted group. Crosslinkers are available, as are pyridyl disulfide crosslinkers. Carbodiimides are a classic example of heterobifunctional crosslinkers, linking a carboxyl to an amine to give an amide bond.

[0432] The subject antibodies, in some embodiments, comprise a "radiopaque" label, e.g., a label that can be easily visualized using X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide salts, bromide salts, or barium salts. Other radiopaque materials are also known, including, but not limited to, organobismuth derivatives, radiopaque multiurethanes, organobismuth composites, radiopaque barium multimer complexes, and the like.

[0433] In some embodiments, the subject antibodies comprise a polyamine modification. The subject antibodies may be modified with either natural or synthetic polyamines. Useful natural polyamines include putrescine, spermidine, spermine, 1,3-deaminopropane, norspermidine, syn-homospermidine, thermine, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines have the empirical formula C X H Y N Zand may be a cyclic or acyclic, branched or unbranched hydrocarbon chain of 3 to 12 carbon atoms, further containing 1 to 6 NR or N(R) groups, where R is H, (C1-C4) alkyl, phenyl, or benzyl. The polyamine can be linked to the antibody using any standard cross-linking method.

[0434] Antibody modification methods The anti-Nectin-4 antibody complexes of the present disclosure can have: 1) an Ig heavy chain constant region conjugated to a substructure of interest; and an Ig light chain constant region conjugated to a substructure of interest; 2) an Ig heavy chain constant region conjugated to a substructure of interest; and an Ig light chain constant region not conjugated to a substructure of interest; or 3) an Ig heavy chain constant region not conjugated to a substructure of interest; and an Ig light chain constant region conjugated to a substructure of interest. The subject anti-Nectin-4 antibody complexes can also include a VH domain and / or a VL domain conjugated to a substructure of interest.

[0435] In one example, an antibody can be modified to contain a 2-formylglycine residue, which can serve as a chemical cue for conjugation of a heterologous moiety. For example, the heavy chain constant region and / or light chain constant region of an anti-Nectin-4 antibody of the present disclosure can be modified to contain an amino acid sequence of a sulfatase motif that can be converted by the action of 2-formylglycine generating enzyme (FGE) to contain 2-formylglycine (fGly). Such a sulfatase motif is sometimes referred to herein as an FGE modification site. The action of FGE is sequence-specific in that it acts on a sulfatase motif located within an immunoglobulin polypeptide. The moiety of interest serves as a component of a reactive partner to react with the aldehyde of the fGly residue of the aldehyde tag after conversion of the tagged Ig polypeptide. A wide range of commercially available reagents can be used to achieve conjugation of the moiety of interest to the fGly residue of the aldehyde-tagged Ig polypeptide. For example, aminooxy, hydrazide, or thiosemicarbazide derivatives of many moieties of interest are suitable reactive partners and are readily available or can be made using standard chemical methods.

[0436] As mentioned above, the amino acid sequence of the anti-Nectin-4 antibody can be converted to a 2-formylglycine (fGly) residue by the action of formylglycine generating enzyme (FGE) in vivo (e.g., during translation of an aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting an aldehyde-tagged protein with FGE in a cell-free system). The FGE-modified fragments may be modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be cleaved. Such sulfatase motifs are sometimes referred to herein as FGE modification sites.

[0437] sulfatase motif The minimum sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, and typically no longer than 6 amino acid residues. Sulfatase motifs provided within Ig polypeptides are at least 5 or 6 amino acid residues in length and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length to define sulfatase motifs that are less than 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acid residues in length.

[0438] In certain embodiments, polypeptides of interest include those in which one or more amino acid residues have been inserted, deleted, or substituted, such as 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 compared to the native amino acid sequence to provide the sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide contains fewer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residue modifications (insertions, additions, deletions, and / or substitutions) of the amino acid sequence compared to the native amino acid sequence of the polypeptide. When the original amino acid sequence of a polypeptide (e.g., an anti-Nectin-4 antibody) contains one or more residues of a desired sulfatase motif, the total number of residue modifications can be reduced, for example, by site-specifically modifying (inserting, adding, deleting, substituting) amino acid residues adjacent to the native amino acid residue to provide the desired sulfatase motif sequence. In certain embodiments, the degree of modification of the native amino acid sequence of the target anti-Nectin-4 polypeptide is minimized, thereby minimizing the number of amino acid residues inserted, deleted, substituted, or added (e.g., at the N-terminus or C-terminus). Minimizing the degree of amino acid sequence modification of the target anti-Nectin-4 polypeptide can minimize the impact such modifications may have on the function and / or structure of the anti-Nectin-4.

[0439] While it should be noted that aldehyde tags of particular interest are those that at least contain a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it will be readily understood that longer aldehyde tags are also contemplated and encompassed by the present disclosure and may be useful in the compositions and methods of the present disclosure. That is, the aldehyde tag may contain a minimal sulfatase motif of five or six residues, or may be longer, containing a minimal sulfatase motif potentially flanked by additional amino acid residues on the N- and / or C-terminal sides of the motif. For example, aldehyde tags of 5 or 6 amino acid residues are contemplated, as are aldehyde tags of longer amino acid sequences of more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, more than 20, or more amino acid residues.

[0440] The aldehyde tag can be located at or near the C-terminus of the Ig heavy chain; for example, the aldehyde tag can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a natural wild-type Ig heavy chain. The aldehyde tag can be located in the CH1 domain of the Ig heavy chain. The aldehyde tag can be located in the CH2 domain of the Ig heavy chain. The aldehyde tag can be present in the CH3 domain of an Ig heavy chain. The aldehyde tag can be present in an Ig light chain constant region, for example, in a kappa light chain constant region or a lambda light chain constant region.

[0441] In certain embodiments, the sulfatase motif used can be described as follows: X 1 Z 1 X 2 Z 2 X 3 Z 3 (SEQ ID NO: 102) (I'), Z 1 is cysteine ​​or serine (sometimes represented as (C / S)); Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), usually A, G, L, V, or I; X 1 is may or may not be present, and if present, may be any amino acid, but is typically an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), and is typically L, M, V, S, or T, more often L, M, S, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, but are typically an aliphatic amino acid, a polar uncharged amino acid, or a sulfur-containing amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C; e.g., S, T, A, V, or G. In one example, the aldehyde tag is of the formula L(C / S)TPSR (SEQ ID NO: 103), e.g., LCTPSR (SEQ ID NO: 104), or LSTPSR (SEQ ID NO: 105). That is, the present disclosure provides an antibody comprising an aldehyde-tagged Ig heavy chain and / or an aldehyde-tagged Ig light chain, wherein the antibody comprises an Ig constant region amino acid sequence of the heavy and / or light chain that contains such a sulfatase motif.

[0442] For example, in some embodiments, the amino acid sequence of the heavy and / or light chain of anti-Nectin-4 is represented by the formula X 1 Z 1 X 2 Z 2 X 3Z 3 may be modified to provide a sequence of at least five amino acids of the formula: Z 1 is cysteine ​​or serine; Z 2 is a proline or alanine residue; Z 3 is an aliphatic amino acid or a basic amino acid; X 1 is either present or absent, and if present, is any amino acid, except when the heterologous sulfatase motif is at the N-terminus of the polypeptide, where X 1 exists; X 2 and X 3 are each, independently of one another, any amino acid; The sequence is located within or adjacent to a solvent-exposed loop region of an Ig constant region, and the sequence is not located at the C-terminus of an Ig heavy chain.

[0443] The sulfatase motif is typically selected so that it is capable of conversion by a selected FGE, e.g., an FGE present in the host cell in which the aldehyde-tagged polypeptide is expressed, or an FGE that will be contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0444] For example, when the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including a human FGE), the sulfatase motif may be of the formula: X 1 CX 2 PX 3 Z 3 (I'') During the ceremony, X 1 may or may not be present, and if present, any amino acid , for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I.

[0445] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: 104), MCTPSR (SEQ ID NO: 105), VCTPSR (SEQ ID NO: 106), LCSPSR (SEQ ID NO: 107), LCAPSR (SEQ ID NO: 108), LCVPSR (SEQ ID NO: 109), LCGPSR (SEQ ID NO: 110), ICTPAR (SEQ ID NO: 111), LCTPSK (SEQ ID NO: 112), MCTPSK (SEQ ID NO: 113), VCTPSK (SEQ ID NO: 114), LCSPSK (SEQ ID NO: 115), LCAPSK (SEQ ID NO: 116), LCVPSK (SEQ ID NO: 117), LCGPSK (SEQ ID NO: 118), LCTPSA (SEQ ID NO: 119), ICTPAA (SEQ ID NO: 120), MCTPSA (SEQ ID NO: 121), VCTPSA (SEQ ID NO: 122), LCSPSA (SEQ ID NO: 123), LCAPSA (SEQ ID NO: 124), LCVPSA ​​(SEQ ID NO: 125), and LCGPSA (SEQ ID NO: 126).

[0446] fGly-containing sequence Typically, the FGE used to promote the conversion of a cysteine ​​or serine in the sulfatase motif of the aldehyde tag of a target polypeptide to fGly is selected depending on the sulfatase motif present in the aldehyde tag. The FGE may be native to the host cell expressing the aldehyde-tagged polypeptide, or the host cell may be genetically modified to express a suitable FGE. In some embodiments, it may be desirable to express the aldehyde-tagged protein using a sulfatase motif compatible with human FGE in human cells expressing FGE or in host cells, typically mammalian cells, genetically modified to express human FGE. Typically, FGEs suitable for use in generating fGly-modified antibodies can be obtained from natural sources or synthetically produced. For example, suitable FGEs can be obtained from biological sources that naturally produce FGE or that have been genetically modified to express a recombinant gene encoding FGE. Numerous FGE-encoding nucleic acids are known in the art and can be readily obtained.

[0447] When FGE acts on the sulfatase motif, Z 1 is oxidized to generate a 2-formylglycine (fGly) residue. Furthermore, upon completion of both the FGE-mediated transformation and the reaction with a reactive partner containing the target substructure, Z in the above formula 1 The fGly position in is covalently attached to a moiety of interest (e.g., a detectable label, a water-soluble polymer, a polypeptide, a drug, an active agent, etc.). That is, the present disclosure provides anti-Nectin-4 antibodies having amino acid sequences modified to include an fGly group.

[0448] When FGE acts on the heavy and / or light chains of anti-nectin-4, the serine or cysteine ​​in the sulfatase motif is modified to fGly. That is, the sulfatase motif containing this fGly can be expressed as follows: X 1 (fGly)X 2 Z 2X 3 Z 3 (SEQ ID NO: 127) (I''') During the ceremony, fGly is a formylglycine residue; Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0449] As described above, to prepare the conjugate, a polypeptide containing an fGly residue may be conjugated to a drug or active agent by reacting fGly with a reactive group of a linker attached to the drug or active agent (e.g., a hydrazinyl-indolyl linking moiety or a hydrazinyl-pyrrolo-pyridinyl linking moiety, as described above) to generate an fGly'-containing sulfatase motif. As used herein, the term fGly' refers to the amino acid residue of a sulfatase motif that is linked to a drug or active agent via a linker, as described herein. That is, the present disclosure provides anti-nectin-4 antibody conjugates (also referred to herein as "anti-nectin-4 conjugates").

[0450] In one embodiment, the anti-nectin-4 conjugate comprises a fGly'-containing sulfatase motif of the formula: X 1 (fGly')X 2 Z 2 X 3 Z 3 (SEQ ID NO: 128) (II) During the ceremony, fGly' is an amino acid residue linked to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1may be present or absent, and if present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0451] In some embodiments, the sequence of formula (II) is located at the C-terminus of the heavy chain constant region of the anti-Nectin-4 antibody. In some cases, the heavy chain constant region comprises the sequence of formula (II): X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue linked to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1may be present or absent, and if present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; The above sequence is C-terminal to the amino acid sequence QKSLSLSPGK (SEQ ID NO: 129), and the above sequence may contain 1, 2, 3, 4, 5, or 5-10 amino acids that are not present in the native wild-type Ig heavy chain constant region.

[0452] In one embodiment, the heavy chain constant region comprises the sequence SLSLSPGSL(fGly')TPSRGS (SEQ ID NO: 248) at the C-terminus of an Ig heavy chain, for example, instead of the native SLSLSPGK (SEQ ID NO: 249) sequence.

[0453] In one embodiment, the heavy chain constant region comprises the sequence SPGSL(fGly')TPSRGS (SEQ ID NO: 130) at the C-terminus of the Ig heavy chain, for example, instead of the native SPGK (SEQ ID NO: 131) sequence.

[0454] In some embodiments, the amino acid residue (fGly') linked to the drug or active agent is located in the light chain constant region of the anti-Nectin-4 antibody. In some embodiments, the light chain constant region comprises the sequence of formula (II): X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue linked to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may or may not be present, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, provided that the sulfatase motif is not present in the target polypeptide. If it is at the N-terminus of the peptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; The sequence is C-terminal to the amino acid sequence KVDNAL (SEQ ID NO: 132) and / or N-terminal to the amino acid sequence QSGNSQ (SEQ ID NO: 133).

[0455] In one embodiment, the light chain constant region comprises the sequence KVDNAL(fGly')TPSRQSGNSQ (SEQ ID NO: 134).

[0456] In some embodiments, the amino acid residue (fGly') linked to the drug or active agent is located in the heavy chain CH1 region of the anti-Nectin-4 antibody. In some embodiments, the heavy chain CH1 region comprises the sequence of formula (II): X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue linked to a drug or active agent via a linker as described herein; Z 2 is either a proline or alanine residue (sometimes represented as (P / A)); Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that if the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; X 2 and X 3 can be, independently of each other, any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; The sequence is C-terminal to the amino acid sequence SWNSGA (SEQ ID NO: 135) and / or N-terminal to the amino acid sequence GVHTFP (SEQ ID NO: 136).

[0457] In one embodiment, the heavy chain CH1 region comprises the sequence SWNSGAL(fGly')TPSRGVHTFP (SEQ ID NO: 137).

[0458] Figure 30A shows a site map indicating potential modification sites for generating aldehyde-tagged Ig polypeptides. The upper sequence is the amino acid sequence of a conserved region of an IgG1 light chain polypeptide (SEQ ID NO: 87), indicating potential modification sites within the Ig light chain; the lower sequence is the amino acid sequence of a conserved region of an Ig heavy chain polypeptide (SEQ ID NO: 88) (GenBank accession number AAG00909), indicating potential modification sites within the Ig heavy chain. The numbering of the heavy and light chains is based on the full-length heavy and light chains.

[0459] Figure 30B shows the modification sites within the immunoglobulin heavy chain that can provide an aldehyde tag. The human immunoglobulin heavy chain constant region of IgG1 (SEQ ID NO: 89; GenBank P01857.1), the human immunoglobulin heavy chain constant region of IgG2 (SEQ ID NO: 90; GenBank P01859.2), and the human immunoglobulin heavy chain constant region of IgG3 (SEQ ID NO: 9 1 shows an alignment of the human immunoglobulin heavy chain constant region of IgG4 (SEQ ID NO: 92; GenBank AAB59394.1), the human immunoglobulin heavy chain constant region of IgA (SEQ ID NO: 93; GenBank AAAT74070). The numbering of the heavy and light chains is based on the complete heavy and light chains.

[0460] Figure 30C shows an alignment of immunoglobulin light chain constant regions, showing possible modification sites within the immunoglobulin light chain to provide an aldehyde tag. Sequence 1 = human kappa light chain constant region; GenBank CAA75031.1; SEQ ID NO: 94. Sequence 2 = human kappa light chain constant region; GenBank BAC0168.1; SEQ ID NO: 95. Sequence 3 = human lambda light chain constant region; GenBank CAA75033; SEQ ID NO: 96. Sequence 4 = Mus musculus light chain constant region; GenBank AAB09710.1; SEQ ID NO: 97. Sequence 5 = Rattus norvegicus light chain constant region; GenBank AAD10133; SEQ ID NO: 98.

[0461] In some embodiments, the sulfatase motif is present at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. The isolated aldehyde-tagged anti-Nectin-4 polypeptide may comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described herein, wherein the sulfatase motif is present within or adjacent to a surface-accessible loop region of the anti-Nectin-4 polypeptide heavy chain constant region.

[0462] Examples of surface-exposed loop regions of IgG1 heavy chains include: 1) ASTKGP (SEQ ID NO: 138); 2) KSTSGGT (SEQ ID NO: 139); 3) PEPV (SEQ ID NO: 140); 4) NSGALTSG (SEQ ID NO: 141); 5) NSGALTSGVHTFPAVLQSSGL (SEQ ID NO: 142); 6) QSSGL (SEQ ID NO: 143); 7) VTV; 8) QTY; 9) TQTY (SEQ ID NO: 144); 10) HKPSN (SEQ ID NO: 145); 11) EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 146); 12) FPPKP (SEQ ID NO: 147); 13) ISRTP (SEQ ID NO: 148); 14) DVSHEDPEV (SEQ ID NO: 149); 15 ) SHEDPEV (SEQ ID NO: 150); 16) DG; 17) DGVEVHNAK (SEQ ID NO: 151); 18) HNA; 19) QYNST (SEQ ID NO: 152); 20) VLTVL (SEQ ID NO: 153); 21) GKE; 22) NKALPAP (SEQ ID NO: 154); 23) SKAKGQPRE (SEQ ID NO: 155); 24) KAKGQPR (SEQ ID NO: 156); 25) PPSRKELTKN (SEQ ID NO: 157); 26) YPSDI (SEQ ID NO: 158); 27) NGQPENN (SEQ ID NO: 159); 28) TPPVLDSDGS (SEQ ID NO: 160); 29) HEALHNHYTQKSLSLSPGK (SEQ ID NO: 161); and 30) SLSPGK (SEQ ID NO: 162).

[0463] Examples of surface-exposed loop regions of IgG2 heavy chains include: 1) ASTKGP (SEQ ID NO: 163); 2) PCSRSTSESTAA (SEQ ID NO: 164); 3) FPEPV (SEQ ID NO: 165); 4) SGALTSGVHTFP (SEQ ID NO: 166); 5) QSSGLY (SEQ ID NO: 167); 6) VTV; 7) TQT; 8) HKP; 9) DK; 10) VAGPS (SEQ ID NO: 168); 11) FPPKP (SEQ ID NO: 169); 12) RTP; 13) DVSHEDPEV (SEQ ID NO: 170); 14) DGV EVHNAK (SEQ ID NO: 171); 15) FN; 16) VLTVV (SEQ ID NO: 172); 17) GKE; 18) NKGLPAP (SEQ ID NO: 173); 19) SKTKGQPRE (SEQ ID NO: 174); 20) PPS; 21) MTKNQ (SEQ ID NO: 175); 22) YPSDI (SEQ ID NO: 176); 23) NGQPENN (SEQ ID NO: 177); 24) TPPMLDSDGS (SEQ ID NO: 178); 25) GNVF (SEQ ID NO: 179); and 26) HEALHNHYTQKSLSLSPGK (SEQ ID NO: 180).

[0464] Examples of surface-exposed loop regions of IgG3 heavy chains include: 1) ASTKGP (SEQ ID NO: 181); 2) PCSRSTSGGT (SEQ ID NO: 182); 3) FPEPV (SEQ ID NO: 183); 4) SGALTSGVHTFPAVLQSSG (SEQ ID NO: 184); 5) V; 6) TQT; 7) HKPSN (SEQ ID NO: 185); 8) RVELKTPLGD (SEQ ID NO: 186); 9) CPRCPKP (SEQ ID NO: 187); 10) PKSCDTPPPCPRCPAPELLGG (SEQ ID NO: 188); 11) FPPKP (SEQ ID NO: 189); 12) RTP; 13) DVSHEDPEV (SEQ ID NO: 190); 14) DGVEVHNAK (SEQ ID NO: 191); 15) YN; 16) VL; 17) GKE; 18) NKALPAP (SEQ ID NO: 192); 19) SKTKGQPRE (SEQ ID NO: 193); 20) PPSREEMTKN (SEQ ID NO: 194); 21) YPSDI (SEQ ID NO: 195); 22) SSGQPENN (SEQ ID NO: 196); 23) TPPMLDSDGS (SEQ ID NO: 197); 24) GNI; 25) HEALHNR (SEQ ID NO: 198); and 26) SLSPGK (SEQ ID NO: 199).

[0465] Examples of surface-exposed loop regions of IgG4 heavy chains include: 1) STKGP (SEQ ID NO: 200); 2) PCSRSTSESTAA (SEQ ID NO: 201); 3) FPEPV (SEQ ID NO: 202); 4) SGALTSGVHTFP (SEQ ID NO: 203); 5) QSSGLY (SEQ ID NO: 204); 6) VTV; 7) TKT; 8) HKP; 9) DK; 10) YG; 11) CPAPEFLGGPS (SEQ ID NO: 205); 12) FPPKP (SEQ ID NO: 206); 13) RTP; 14) DVSQEDPEV (SEQ ID NO: 207); 15) DGVEVHNAK (SEQ ID NO: 208); 16) FN; 17) VL; 18) GKE; 19) NKGLPSS (SEQ ID NO: 209); 20) SKAKGQPREP (SEQ ID NO: 210); 21) PPSQEEMTKN (SEQ ID NO: 211); 22) YPSDI (SEQ ID NO: 212); 23) NG; 24) NN; 25) TPPVLDSDGS (SEQ ID NO: 213); 26) GNVF (SEQ ID NO: 214); and 27) HEALHNHYTQKSLSLSLGK (SEQ ID NO: 215).

[0466] Examples of surface-exposed loop regions of IgA heavy chains include: 1) ASPTSPKVFPLSL (SEQ ID NO: 216); 2) QPDGN (SEQ ID NO: 217); 3) VQGFFPQEPL (SEQ ID NO: 218); 4) SGQGVTARNFP (SEQ ID NO: 219); 5) SGDLYTT (SEQ ID NO: 220); 6) PATQ (SEQ ID NO: 221); 7) GKS; 8) YT; 9) CHP; 10) HRPA (SEQ ID NO: 222); 11) LLGSE (SEQ ID NO: 223); 12) GLRDASGV (SEQ ID NO: 224) ); 13) SSGKSAVQGP (SEQ ID NO: 225); 14) GCYS (SEQ ID NO: 226); 15) CAEP (SEQ ID NO: 227); 16) PE; 17) SGNTFRPEVHLLPPPSEELALNEL (SEQ ID NO: 228); 18) ARGFS (SEQ ID NO: 229); 19) QGSQELPREKY (SEQ ID NO: 230); 20) AV; 21) AAED (SEQ ID NO: 231); 22) HEAL (SEQ ID NO: 232); and 23) IDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 233).

[0467] Examples of surface-exposed loop regions of Ig light chains (e.g., human kappa light chains) include: 1) RTVAAP (sequence number 234); 2) PPS; 3) Gly (see, e.g., the Gly at position 150 of the human kappa light chain sequence shown in Figure 8C); 4) YPREA (sequence number 235); 5) PREA (sequence number 236); 6) DNALQSGN (sequence number 237); 7) TEQDSKDST (sequence number 238); 8) HK; 9) HQGLSS (sequence number 239); and 10) RGEC (sequence number 240).

[0468] Examples of surface-exposed loop regions of Ig λ light chains include: QPKAAP (SEQ ID NO: 241), PPS, NK, DFYPGAV (SEQ ID NO: 242), DSSPVKAG (SEQ ID NO: 243), TTP, SN, HKS, EG, and APTECS (SEQ ID NO: 244).

[0469] The constant region of the HC of the anti-Nectin-4 antibody disclosed herein may be selected from one of the following sequences:

[0470] [Table 6]

[0471] In SEQ ID NO: 70, the italicized residues at the C-terminus of the heavy chain constant region replace the lysine residue at the C-terminus of a standard IgG1 heavy chain. The bolded italicized residue (LCTPSR (SEQ ID NO: 104)) constitutes an aldehyde tag, and this C is converted to an fGly residue by FGE after expression of the heavy chain to generate L(fGly)TPSR (SEQ ID NO: 245). This fGly is modified to fGly' to generate L(fGly')TPSR (SEQ ID NO: 246). fGly' refers to the amino acid residue of the anti-Nectin-4 antibody that is linked to the moiety of interest (e.g., a drug). The non-bolded italicized residues are additional residues that differ from the standard IgG1 heavy chain sequence.

[0472]

Table 7

[0473]

Table 8

[0474]

Table 9

[0475]

Table 10

[0476]

Table 11

[0477]

Table 12

[0478]

Table 13

[0479]

Table 14

[0480]

Table 15

[0481] Table 16

[0482] Table 17

[0483] [Table 18]

[0484] [Table 19]

[0485] [Table 20]

[0486] [Table 21]

[0487] [Table 22]

[0488] The bolded residues (LCTPSR) constitute an aldehyde tag, and this C is converted to a fGly residue by FGE after expression of the heavy chain. This fGly may be converted to fGly', which refers to the amino acid residue of the anti-Nectin-4 antibody that is linked to the moiety of interest (e.g., a drug).

[0489] Drugs and Active Agents In some cases, the anti-Nectin-4 antibodies of the present disclosure may comprise a drug or active agent covalently linked to the heavy and / or light chain of the antibody (e.g., the W chain of the conjugate of Formula (I) described herein). 1 For example, the antibody conjugates of the present disclosure have a substituent W 1 may contain a drug or active agent as the substituent W 2The "antibody" may include a second drug or active agent. Any of a number of drugs are suitable for use, or can be modified to be suitable for use, as a reactive partner for conjugation to an antibody. "Drugs" include small molecule drugs, peptidic drugs, toxins (e.g., cytotoxins), and the like.

[0490] "Small molecule drug," as used herein, refers to a compound, e.g., an organic compound, that exhibits a pharmaceutical activity of interest and typically has a molecular weight of about 800 Da or less, or 2000 Da or less, but can encompass molecules up to 5 kDa and can be as large as about 10 kDa. Inorganic small molecules refer to molecules that contain no carbon atoms, while organic small molecules refer to compounds that contain at least one carbon atom.

[0491] In certain embodiments, the drug or active agent may be maytansine. "Maytansine," "maytansine moiety," "maytansine active agent moiety," and "maytansinoid" refer to maytansine, as well as analogs and derivatives thereof, and pharmaceutically active maytansine moieties and / or portions thereof. The maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties, including, but not limited to, maytansine and its analogs and derivatives (e.g., deacylmaytansine) as described herein.

[0492] In certain embodiments, the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and / or portion thereof. The auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties, including, but not limited to, auristatin and its analogs and derivatives as described herein. Examples of agents useful in the described conjugates and compounds include, but are not limited to, auristatins or auristatin derivatives, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like.

[0493] In certain embodiments, the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and / or portion thereof. The duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties, including, but not limited to, duocarmycin and its analogs and derivatives as described herein. Examples of drugs useful in the conjugates and compounds described herein include, but are not limited to, duocarmycins or duocarmycin derivatives, such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, and derivatives thereof. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bisceresin, or carzelesin.

[0494] In certain embodiments, the drug or active agent may be a topoisomerase inhibitor, such as camptothecin, or an analog or derivative thereof, or a pharmaceutically active camptothecin moiety and / or portion thereof. The camptothecin conjugated to the subject antibodies can be any of a variety of camptothecin moieties, including, but not limited to, camptothecin and its analogs and derivatives as described herein. Examples of drugs useful in the conjugates described herein include, but are not limited to, camptothecin or camptothecin derivatives, such as SN-38, belotecan, exatecan, 9-aminocamptothecin (9-AC), derivatives thereof, and the like.

[0495] In certain embodiments, the drugs or active agents of Formula (I) and Formula (II) described herein (e.g., W 1 and / or W 2 ) is camptothecine, or an analog or derivative thereof. For example, in some cases, the camptothecine, or analog or derivative thereof is a compound of formula (III):

[0496] [ka] (III)

[0497] During the ceremony, R 31 and R 32 are each, independently of one another, hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, aryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 31 and R 32 are optionally cyclically linked to form a 5- or 6-membered cycloalkyl or heterocyclyl ring; R 33 and R 34 are each, independently of one another, selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 33 and R 34 are optionally cyclically linked to form a 5- or 6-membered cycloalkyl or heterocyclyl ring; R 35 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 36 is OH and OC(O)R 37 Selected from; R 37 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; It is a compound of formula (III).

[0498] In some embodiments of Formula (III), the linker L in Formula (I) or Formula (II) A is R of the compound of formula (III) 31 , R 32 , R 33, R 34 , R 35 , or R 36 In some embodiments of Formula (III), the linker L in Formula (I) or Formula (II) is B is R of the compound of formula (III) 31 , R 32 , R 33 , R 34 , R 35 , or R 36 is bonded to.

[0499] In one embodiment, R 31 and R 32 are each, independently of one another, selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 31 and R 32 are optionally linked cyclically to form a 5- or 6-membered cycloalkyl or heterocyclyl ring.

[0500] In one embodiment, R 31 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 31 is hydrogen. In some embodiments, R 31 is halogen (e.g., F, Cl, Br, I). In some embodiments, R 31 is hydroxy. In one embodiment, R 31 is amino or substituted amino. In one embodiment, R 31 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 31 is methyl. In one embodiment, R 31 is a C2-substituted alkyl, such as —CHCHNH(CH(CH)). In certain embodiments, R 31 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 Alkenyl or substituted alkenyl, such as substituted alkenyl In one embodiment, R 31 is alkynyl or substituted alkynyl. In some embodiments, R 31 is alkoxy or substituted alkoxy. In some embodiments, R 31 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 31 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 31 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 31is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0501] In one embodiment, R 32 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 32 is hydrogen. In some embodiments, R 32 is halogen (e.g., F, Cl, Br, I). In some embodiments, R 32 is hydroxy. In one embodiment, R 32 is amino or substituted amino. In one embodiment, R 32 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 32 is methyl. In one embodiment, R 32 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 32 is alkynyl or substituted alkynyl. In some embodiments, R 32 is alkoxy or substituted alkoxy. In some embodiments, R32 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 32 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 32 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 32 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0502] In one embodiment, R 31 and R 32 are optionally cyclically linked to form a 5- or 6-membered cycloalkyl or heterocyclyl ring. 31 and R 32 are linked in a ring to form a 5- or 6-membered cycloalkyl. 31 and R 32 are joined in a ring to form a 5- or 6-membered heterocyclyl. 31 and R 32 are linked in a ring to form a 5-membered cycloalkyl.31 and R 32 are linked in a ring to form a 6-membered cycloalkyl. 31 and R 3 2 are linked in a ring to form a 5-membered heterocyclyl. 31 and R 32 are joined together to form a six-membered heterocyclyl.

[0503] In one embodiment, R 33 and R 34 are each, independently of one another, selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 33 and R 34 are optionally cyclically linked to form a 5- or 6-membered cycloalkyl or heterocyclyl ring.

[0504] In one embodiment, R 33 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 33 is hydrogen. In some embodiments, R 33 is halogen (e.g., F, Cl, Br, I). In some embodiments, R 33 is hydroxy. In one embodiment, R 33 is amino or substituted amino. In one embodiment, R 33 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 33 is methyl. In one embodiment, R 33 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 33 is alkynyl or substituted alkynyl. In some embodiments, R 33 is alkoxy or substituted alkoxy. In some embodiments, R 33 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 33 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 33 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 33 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0505] In one embodiment, R 34 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 34 is hydrogen. In some embodiments, R 34 is halogen (e.g., F, Cl, Br, I). In some embodiments, R 34 is hydroxy. In one embodiment, R 34 is amino or substituted amino. In one embodiment, R 34 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 34 is methyl. In one embodiment, R 34 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl , or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 34 is alkynyl or substituted alkynyl. In some embodiments, R 34 is alkoxy or substituted alkoxy. In some embodiments, R 34 is aryl or substituted aryl, e.g., C 5~8 Aryl or C5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 34 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 34 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 34 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0506] In one embodiment, R 33 and R 34 are optionally cyclically linked to form a 5- or 6-membered cycloalkyl or heterocyclyl ring. 33 and R 34 are linked in a ring to form a 5- or 6-membered cycloalkyl. 33 and R 34 are joined in a ring to form a 5- or 6-membered heterocyclyl. 33 and R 34 are linked in a ring to form a 5-membered cycloalkyl. 33 and R 34are linked in a ring to form a 6-membered cycloalkyl. 33 and R 34 are linked in a ring to form a 5-membered heterocyclyl. 33 and R 34 are joined together to form a six-membered heterocyclyl.

[0507] In one embodiment, R 35 is selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 35 is hydrogen. In some embodiments, R 35 is halogen (e.g., F, Cl, Br, I). In some embodiments, R 35 is hydroxy. In one embodiment, R 35 is amino or substituted amino. In one embodiment, R 35 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 35 is methyl. In one embodiment, R 35 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 35 is alkynyl or substituted alkynyl. In some embodiments, R 35is alkoxy or substituted alkoxy. In some embodiments, R 35 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 35 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 35 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~ 5 cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 35 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0508] In one embodiment, R 36 is OH and OC(O)R 37 In one embodiment, R 36 is OH. In some embodiments, R 36 is OC(O)R 37 is.

[0509] In one embodiment, R 37is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 37 is hydrogen. In some embodiments, R 37 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 37 is C 2~6 Alkenyl or C 2~6 substituted alkenyl, or C 2~4 Alkenyl or C 2~4 substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In some embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 37 is alkynyl or substituted alkynyl. In some embodiments, R 37 is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 37 is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 37 is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 37 is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl and the like.

[0510] In certain embodiments, the compound of Formula (III) has the structure of Formula (IIIa):

[0511] [ka] (IIIa)

[0512] In certain embodiments of the compound of Formula (IIIa), R 33 is as above.

[0513] In certain embodiments of the compound of Formula (IIIa), R 36 is as above.

[0514] In certain embodiments of the compound of Formula (IIIa), R 33 is OH, and the linker L A or L B R 36 In certain embodiments of the compound of Formula (IIIa), the linker L A or L B R 33 is bonded to R 36 is OH.

[0515] In certain embodiments, the compound of Formula (III) has the structure of Formula (IIIb):

[0516] [ka] (IIIb)

[0517] In certain embodiments of the compound of Formula (IIIb), R 31a is selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl. 31a is hydrogen. In some embodiments, R 31a is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 31a is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 31a is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 31a is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 31a is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl, etc. In some embodiments, R 31a is carboxyl. In one embodiment, R 31a is a carboxyl ester. In one embodiment, R 31a is acyl. In one embodiment, R 31a is sulfonyl.

[0518] In certain embodiments of the compound of Formula (IIIb), R 36 is as above.

[0519] In certain embodiments of the compound of Formula (IIIb), R 31a is selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl; and the linker L A or L B R 36 In certain embodiments of the compound of Formula (IIIb), the linker L A or L B R 31a is bonded to R 36 is OH.

[0520] In certain embodiments, the compound of Formula (III) has the structure of Formula (IIIc):

[0521] [ka] (IIIc)

[0522] In certain embodiments of the compound of Formula (IIIc), R 31bis selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl. 31b is hydrogen. In some embodiments, R 31b is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 31b is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 31b is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 31b is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 31b is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl, etc. In some embodiments, R31b is carboxyl. In one embodiment, R 31b is a carboxyl ester. In one embodiment, R 31b is acyl. In one embodiment, R 31b is sulfonyl.

[0523] In certain embodiments of the compound of Formula (IIIc), R 36 is as above.

[0524] In certain embodiments of the compound of Formula (IIIc), R 31b is selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl; and the linker L A or L B R 36 In certain embodiments of the compound of Formula (IIIc), the linker L A or L B R 31b is bonded to R 36 is OH.

[0525] In certain embodiments, the compound of Formula (III) has the structure of Formula (IIId):

[0526] [ka] (IIId)

[0527] In certain embodiments of compounds of Formula (IIId), R 32a and R 32b are each, independently of the other, selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl.

[0528] In certain embodiments of compounds of Formula (IIId), R 32a is selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl. 32a is hydrogen. In some embodiments, R 32a is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 32a is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 32a is heteroaryl or substituted heteroaryl, e.g., C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 32a is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 32a is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5Heterocyclyl or C 3~5 substituted heterocyclyl, etc. In some embodiments, R 32a is carboxyl. In one embodiment, R 32a is a carboxyl ester. In one embodiment, R 32a is acyl. In one embodiment, R 32a is sulfonyl.

[0529] In certain embodiments of compounds of Formula (IIId), R 32b is selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl. 32b is hydrogen. In some embodiments, R 32b is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In some embodiments, R 32b is aryl or substituted aryl, e.g., C 5~8 Aryl or C 5~8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In some embodiments, R 32b Hetero ants C 5~8 Heteroaryl or C 5~8 In some embodiments, R is a substituted heteroaryl, such as a C5 heteroaryl or a C5 substituted heteroaryl, or a C6 heteroaryl or a C6 substituted heteroaryl. 32b is cycloalkyl or substituted cycloalkyl, e.g., C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6Cycloalkyl or C 3~6 substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 substituted cycloalkyl, etc. In some embodiments, R 32b is heterocyclyl or substituted heterocyclyl, e.g., C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 substituted heterocyclyl, etc. In some embodiments, R 32b is carboxyl. In one embodiment, R 32b is a carboxyl ester. In one embodiment, R 32b is acyl. In one embodiment, R 32b is sulfonyl.

[0530] In certain embodiments of compounds of Formula (IIId), R 36 is as above.

[0531] In certain embodiments of compounds of Formula (IIId), R 32a and R 32b are each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl; and the linker L A or L B R 36 In certain embodiments of the compound of Formula (IIId), the linker L A or L B R 32a or R 32b is bonded to R 36 is OH. In certain embodiments of the compound of Formula (IIId), the linker L A or L B R 32a is bonded to R 36 is OH. In certain embodiments of the compound of Formula (IIId), the linker L Aor L B R 32b is bonded to R 36 is OH.

[0532] In certain embodiments, the agent is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulant, a Toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.

[0533] For example, a cytotoxin can include any compound that results in cell death (eg, necrosis or apoptosis) or a decrease in cell viability.

[0534] Kinase inhibitors include, but are not limited to, adavosertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitinib, tucatinib, vandetanib, and vemurafenib.

[0535] For example, selective estrogen receptor modulators include, but are not limited to, endoxifen, tamoxifen, afimoxifen, toremifene, and the like.

[0536] Immunostimulants can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony-stimulating factors, interferons, interleukins, etc. TLR agonists include, but are not limited to, imiquimod, resiquimod, etc.

[0537] Oligonucleotide drugs include fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, and voraneso. These include, but are not limited to, lucene, inotersen, tofersen, and tominersen.

[0538] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like.

[0539] Cytokines include albinterferon α-2B, aldesleukin, ALT-801, anakinra, ancestim, avotermin, valgrastim, bempegaldesleukin, binetrakin, syntredekin besudotox, CTCE-0214, darbepoetin alfa, denileukin diftitox, dulanermin, eddekin α, emfilermin, epoetin δ, erythropoietin, and human interleukin. Interferon-2, interferon alpha, interferon alpha-2c, interferon alpha-n1, interferon alpha-n3, interferon alfacon-1, interferon beta-1a, interferon beta-1b, interferon gamma-1b, interferon kappa, interleukin-1α, interleukin-10, interleukin-7, lenograstim, religistim, lipegfilgrastim, lorukafusp alpha alfa), Maxy-G34, methoxypolyethylene glycol-epoetin beta, molgramostim, muprestim, nagressipene, oprelvekin, pegfilgrastim, pegylidecaquin, peginterferon alfa-2a, peginterferon alfa-2b, peginterferon beta-1a, peginterferon lambda-1a, recombinant CD40-ligand, regramostim, romiplostim, sargramostim, thrombopoietin, tucotuzumab celmoleukin, viral macrophage inflammatory protein, and the like.

[0540] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.

[0541] The term "peptide drug," as used herein, refers to an amino acid-containing polymeric compound and is intended to encompass natural and non-natural peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptidomimetics. These peptide drugs may be obtained by chemical synthesis or may be generated from genetically engineered sources (e.g., recombinant sources). Peptide drugs may range in molecular weight from 200 Da to 10 kDa or more. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; antiangiogenic peptides; peptides that activate B cells; peptides that activate T cells; antiviral peptides; peptides that inhibit viral fusion; peptides that increase the production of one or more lymphocyte populations; antimicrobial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti-inflammatory peptides; peptides that regulate glucose metabolism; antithrombotic peptides; antinociceptive peptides; vasodilatory peptides; platelet aggregation inhibitors; analgesics; and the like.

[0542] Further examples of agents useful in the conjugates and compounds described herein include, but are not limited to, Tubulysin M, calicheamicin, STAT3 inhibitors, α-amanitin, aurora kinase inhibitors, belotecan, and anthracyclines.

[0543] In some cases, the agent is a toxin, e.g., a cytotoxin. Ribosome-inactivating proteins (RIPs), a class of proteins ubiquitous in higher plants, are known to inhibit the action of such cells. Examples of cytotoxins include, but are not limited to, ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxins (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pokeweed antiviral protein (PAP), botulinum toxin, bryodin, momordin, and bouganin.

[0544] In some cases, the drug is a cancer chemotherapeutic agent.Cancer chemotherapeutic agents include non-peptide (e.g., non-proteinaceous) compounds that reduce the proliferation of cancer cells, including cytotoxic drugs and cytostatics.Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, anticancer antibiotics, plant (vinca) alkaloids, and steroid hormones.Peptide compounds can also be used.

[0545] Suitable cancer chemotherapeutic agents include dolastatins, and their active analogs and derivatives; and auristatins, and their active analogs and derivatives. Suitable cancer chemotherapeutic agents also include maytansinoids, and their active analogs and derivatives; and duocarmycins, and their active analogs and derivatives.

[0546] Drugs that act to reduce cell proliferation are known in the art and are widely used.Such drugs include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including but not limited to mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, dacarbazine, and temozolomide.

[0547] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0548] Suitable natural products and derivatives thereof (e.g., vinca alkaloids, anticancer antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins such as etoposide and teniposide, and the like; antibiotics such as ant...

Claims

1. Anti-Nectin-4 antibodies, including: A heavy chain variable region (VH) chain comprising heavy chain CDRs 1-3 (HCDRs 1-3) of the VH chain having a sequence selected from SEQ ID NOs: 1-17; and A light chain variable region (VL) chain comprising light chain CDRs 1-3 (LCDRs 1-3) of the VL chain having a sequence selected from SEQ ID NOs: 18-31.

2. the VH chain comprises HCDRs 1-3 of a VH chain having a sequence selected from SEQ ID NOs: 1-6; The anti-Nectin-4 antibody of claim 1, wherein the VL chain comprises LCDR1 to LCDR3 of a VL chain having a sequence selected from SEQ ID NOs: 18 to 23.

3. the VH chain comprises HCDR1-3 of the VH chain having the sequence of SEQ ID NO:5; The anti-Nectin-4 antibody of claim 2, wherein the VL chain comprises LCDR1 to LCDR3 of a VL chain having the sequence of SEQ ID NO: 19 or 23.

4. The anti-Nectin-4 antibody according to claim 1, wherein the anti-Nectin-4 antibody is an IgG1 antibody.

5. A conjugate of formula (I) 【Chemical 1】 (I) During the ceremony, Z １ , Z ２ , Z ３ , and Z ４ are each independently of each other, CR ４ , N, and C-L Ｂ -W ２ Selected from: R １ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R ２ and R ３ are each, independently of one another, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R ２ and R ３ are optionally joined in a ring to form a 5- or 6-membered heterocyclyl; Each R ４ are each independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L Ａ is the first linker; L Ｂ is a second linker; W １ is the first agent; W ２ is the second agent; W ３ is the anti-Nectin-4 antibody according to claim 1. A conjugate of formula (I).

6. The conjugate of claim 5 , wherein the first linker and / or the second linker comprises a glycoside.

7. 7. The conjugate of claim 6, wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

8. The conjugate of claim 5, selected from the following: 【Chemistry 2】 、 【Chemistry 3】 , and 【Chemistry 4】 。

9. The anti-Nectin-4 antibody comprises a sequence of formula (II): X １ (fGly')X ２ Z ２ X ３ Z ３ (SEQ ID NO: 128) (II), During the ceremony, X １ may be present or absent, and if present, may be any amino acid, provided that when said sequence is at the N-terminus of said complex, X １ exists; fGly' is linked to the first drug via the first linker or the second linker, respectively. an amino acid residue linking the α- or β-acting agent to the second agent; X ２ and X ３ are each, independently of one another, any amino acid; Z ２ is either a proline or an alanine residue; Z ３ is a basic amino acid or an aliphatic amino acid, The complex of claim 5.

10. 10. The conjugate of claim 9, wherein the sequence of formula (II) is L(fGly')TPSR (SEQ ID NO: 246).

11. The conjugate of claim 9, wherein the sequence of formula (II) is located at any of the following positions in the heavy chain constant region of the anti-Nectin-4 antibody: the C-terminus set forth in SEQ ID NO: 70; before the 58th Q residue set forth in SEQ ID NO: 71, 75, 79, or 83; before the 61st G residue set forth in SEQ ID NO: 72, 76, 80, or 84; before the 91st N residue set forth in SEQ ID NO: 73, 77, 81, or 85; before the 116th E residue set forth in SEQ ID NO: 74, 78, 82, or 86; on the C-terminal side of the amino acid sequence SWNSGA (SEQ ID NO: 135) in the CH1 region, or on the N-terminal side of the amino acid sequence GVHTFP (SEQ ID NO: 136) in the CH1 region.

12. The conjugate of claim 9, wherein the sequence of formula (II) is located at any of the following positions in the heavy chain constant region of the anti-Nectin-4 antibody: the C-terminus set forth in SEQ ID NO: 70 and the C-terminal side of the amino acid sequence SWNSGA (SEQ ID NO: 135) in the CH1 region, or the N-terminal side of the amino acid sequence GVHTFP (SEQ ID NO: 136) in the CH1 region; or before the 91st N residue set forth in SEQ ID NO: 73, 77, 81, or 85 and before the 116th E residue set forth in SEQ ID NO: 74, 78, 82, or 86.

13. An antibody according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 12; and pharmaceutically acceptable excipients, A pharmaceutical composition comprising:

14. The pharmaceutical composition of claim 13 for treating cancer.

15. The pharmaceutical composition of claim 14, wherein the cancer is ovarian cancer, breast ductal carcinoma, lung adenocarcinoma, or pancreatic cancer.

16. The pharmaceutical composition of claim 14, wherein the cancer is characterized by cancer cells that express Nectin-4.

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