CN118043328A

CN118043328A - Antibodies and antibody conjugates specific for NECTIN-4 and methods of use thereof

Info

Publication number: CN118043328A
Application number: CN202280066532.8A
Authority: CN
Inventors: D·杨; M·保宗; F·张; S·丘普拉科夫; Y·C·金; R·M·巴菲尔德; P·M·德雷克
Original assignee: RP Scherer Technologies LLC
Current assignee: RP Scherer Technologies LLC
Priority date: 2021-07-30
Filing date: 2022-07-28
Publication date: 2024-05-14

Abstract

The present disclosure provides antibodies specific for Nectin-4 and antibody conjugates, such as antibody-drug conjugates (ADCs), comprising such antibodies. Methods of producing such antibodies and antibody conjugates, and methods of using the same, are also encompassed by the present disclosure. Also provided are compositions, including in some cases pharmaceutical compositions, comprising the antibodies and antibody conjugates of the disclosure. In certain aspects, methods of using ADCs are provided that include administering to an individual having a cell proliferative disorder a therapeutically effective amount of an antibody or antibody conjugate of the disclosure.

Description

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

Cross Reference to Related Applications

The disclosure of each of U.S. provisional application number 63/227,666 filed on 7-month 30 of 2021, U.S. provisional application number 63/322,914 filed on 3-month 23 of 2022, and U.S. provisional application number 63/344,932 filed on 5-month 23 of 2022, the disclosures of each of which are incorporated herein by reference, are claimed for priority.

Introduction to the invention

Great progress has been made in the field of protein-small molecule therapeutic conjugates, providing a number of clinically beneficial agents, and hopefully more in the coming years. Protein-conjugate therapeutics can provide a number of advantages, resulting in fewer side effects due to, for example, specificity, functional diversity, and relatively low off-target activity. Chemical modification of proteins can extend these advantages by making them more efficient, stable or multi-modal.

Many standard chemical transformations are commonly used to produce and manipulate post-translational modifications of proteins. There are various methods available for selectively modifying the side chains of certain amino acids. For example, carboxylic acid side chains (aspartate and glutamate) can be targeted by initial activation with a water-soluble carbodiimide reagent and subsequent reaction with an amine. Similarly, lysine can be targeted by using an activated ester or isothiocyanate, and cysteine thiols can be targeted using maleimide and a-halo-carbonyl.

One significant obstacle to creating chemically altered protein therapeutics or agents is the production of proteins in homogeneous form that are biologically active. Conjugation of a drug or detectable label to a polypeptide can be difficult to control, resulting in a heterogeneous mixture of conjugates with varying numbers of drug molecules attached and positions of chemical conjugation. In some cases, it may be desirable to use synthetic organic chemical means to control the conjugation site and/or drug or detectable label to the polypeptide to direct precise and selective formation of chemical bonds on the polypeptide.

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 is a member of the adhesion protein Nectin family. The adhesion proteins of the Nectin family are structurally related and exhibit three conserved immunoglobulin-like domains in their extracellular regions (V, C and C). Nectin-4 has a molecular weight of about 55kDa, wherein the extracellular domain has a molecular weight of about 36kDa.

Nectin-4 can form homodimers or heterodimers with Nectin-1. Nectin-4 regulates a variety of cellular activities such as motility, proliferation, differentiation, polarization, and viral entry. Although other Nectin family members are widely expressed in adult tissues, nectin-4 is primarily limited to embryos and placenta. Furthermore, nectin-4 is overexpressed in a variety of solid tumors, such as ovarian, ductal breast, lung adenocarcinoma, and pancreatic cancer. Nectin-4 arises with metastasis associated proteins and can be associated with disease progression and poor prognosis.

Summary of the inventionsummary

The present disclosure provides antibodies specific for Nectin-4 and antibody conjugates (e.g., antibody-drug conjugates (ADCs)) comprising such antibodies. Methods of producing such antibodies and antibody conjugates, and methods of using the same, are also encompassed by the present disclosure. Each of the embodiments is described in more detail in the following sections. Also provided are compositions, including in some cases pharmaceutical compositions, comprising the antibodies of the present disclosure and ADCs. In certain aspects, methods of using an ADC are provided that include administering a therapeutically effective amount of an ADC of the present disclosure to an individual having a cell proliferative disorder.

Brief description of the sequence

SEQ ID NOS 1 to 17: heavy chains of antibodies disclosed herein.

SEQ ID NOS 18 to 31: the light chain of the antibodies disclosed herein.

SEQ ID NOS 32 to 69: CDRs of heavy and light chains of antibodies disclosed herein.

SEQ ID NOS 70 to 86: the heavy chain constant region of the antibodies disclosed herein.

SEQ ID NOS 87 and 88: igG1 heavy and light chains.

SEQ ID NOS 89 to 93: heavy chain constant regions of different Ig isotypes.

SEQ ID NOS: 94 to 98: light chain constant regions of different types and organisms.

SEQ ID NO. 99: human Nectin-4 protein sequence.

SEQ ID NOS: 100 to 101: flexible linkers comprising glycine polymers.

SEQ ID NOS 102 to 126: examples of sulfatase motifs prior to conversion with Formylglycine Generating Enzyme (FGE).

SEQ ID NOS 127 to 128 and 245 to 246: examples of sulfatase motifs after transformation with FGE.

SEQ ID NO. 129: amino acid sequence of the heavy chain constant region of the insertion sulfatase motif.

SEQ ID NOS 130 to 244: sequences within the constant region of different immunoglobulins.

Brief description of the drawings

FIG. 1 shows chimeric anti-Nectin-4 antibodies binding to recombinant human Nectin-4 protein.

FIG. 2 shows chimeric anti-Nectin-4 antibodies binding to recombinant human Nectin-4 protein.

FIG. 3 shows anti-Nectin-4 antibodies binding to human Nectin and Necl family members.

FIG. 4 shows a cloned variant of the 12E11 antibody that binds to human Nectin and Necl family members.

FIG. 5 shows a cloned variant of the 12E11 antibody that binds to human Nectin and NecI family members.

FIG. 6 shows humanized 5D9 variants that bind to human Nectin-4.

FIG. 7 shows humanized 5D9 variants that bind to human Nectin-4.

FIG. 8 shows humanized 5D9 variants that bind to human Nectin-4.

FIG. 9 shows humanized 5D9 variants that bind to human Nectin-4.

FIG. 10 shows the in vitro efficacy of chimeric anti-Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 11 shows the in vitro efficacy of chimeric anti-Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 12 shows the in vitro efficacy of chimeric anti-Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 13 shows the in vitro efficacy of chimeric anti-Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

Figure 14 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

Figure 15 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against SK-BR-3 cells.

FIG. 16 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against MDA-MB-468 cells.

Figure 17 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 18 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against SK-BR-3 cells.

Figure 19 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against MDA-MB-468 cells.

Figure 20 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 21 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against SK-BR-3 cells.

Figure 22 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against MDA-MB-468 cells.

FIG. 23 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 24 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against SK-BR-3 cells.

FIG. 25 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against MDA-MB-468 cells.

Figure 26 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against HEK cells overexpressing human Nectin-4.

FIG. 27 shows the in vitro efficacy of the h5D9 variant against Nectin-4 ADC or control against SK-BR-3 cells.

FIG. 28 shows the in vitro efficacy of humanized 5D9 variant against Nectin-4 ADC or control against MDA-MB-468 cells.

FIG. 29 shows the in vivo efficacy of a double labeled aldehyde-labeled enrolment monoclonal antibody (Enfortumab) conjugated to compound 20 against NCI-H1781 xenograft models.

FIG. 30A shows a map of sites showing possible modification sites for the production of aldehyde-tagged Ig polypeptides. The upper sequence is the amino acid sequence of the conserved region of the IgG1 light chain polypeptide (SEQ ID NO: 87) and shows possible modification sites in the Ig light chain; the lower sequence is the amino acid sequence efficiency of the conserved region of the Ig heavy chain polypeptide (GenBank accession No. AAG 00909) and shows possible modification sites in the Ig heavy chain. Heavy and light chain numbering is based on full length heavy and light chains.

FIG. 30B depicts an alignment of the heavy chain constant regions of the human immunoglobulin of IgG1(SEQ ID NO:89;GenBank P01857.1)、IgG2(SEQ ID NO:90;GenBank P01859.2)、IgG3(SEQ ID NO:91;GenBank P01860.2)、IgG4(SEQ ID NO:92;GenBank AAB59394.1) and IgA (SEQ ID NO:93;GenBank AAAT74070), which shows that modification sites of the aldehyde tag can be provided in the heavy chain of the immunoglobulin. Heavy and light chain numbering is based on full length heavy and light chains.

FIG. 30C depicts an alignment of immunoglobulin light chain constant regions, which shows modification sites that can provide an aldehyde tag in an immunoglobulin light chain. Seq1=homo sapiens kappa light chain constant region; genBank CAA75031.1; SEQ ID NO. 94.Seq2 = homo sapiens kappa light chain constant region; genBank BAC0168.1; SEQ ID NO. 95. Seq3=homo sapiens lambda light chain constant region; genBank CAA75033; SEQ ID NO. 96.Seq4 = mouse light chain constant region; genBank AAB09710.1; SEQ ID NO. 97.Seq5 = brown mouse light chain constant region; genBank AAD10133; SEQ ID NO. 98.

FIG. 30D depicts an alignment of immunoglobulin light chain constant regions, which shows that modification sites can provide aldehyde tags in immunoglobulin light chains. Seq1=homo sapiens kappa light chain constant region; genBank CAA75031.1; SEQ ID NO. 52.Seq2 = homo sapiens kappa light chain constant region; genBank BAC0168.1; SEQ ID NO. 53. Seq3=homo sapiens lambda light chain constant region; genBank CAA75033; SEQ ID NO. 54.Seq4 = mouse light chain constant region; genBank AAB09710.1; SEQ ID NO. 55.Seq5 = brown mouse light chain constant region; genBank AAD10133; SEQ ID NO. 56.

FIG. 31 shows a graph of NCI-H1781 xenograft studies using single 2.5 or 7.5mg/kg intravenous doses of the listed anti-nectin-4 ADCs on day 0. Both VH4/VL1 compound 8 (RED-601) and VH4/VL5 compound 8 use an internal 91N tag and deliver half the loading dose compared to Padcev. Isotype control ADC had minimal activity.

FIG. 32 shows a graph of NCI-H1781 xenograft studies on day 0 using single 2.5 or 7.5mg/kg intravenous doses of either the listed anti-nectin-4 or isotype control ADCs. Preparation of DAR4 format VH4/VL1 compound 25 using a 91N tag (RED-694) and preparation of DAR8 format VH4/VL1 compound 25 using a 91N/116E ditag combination (RED-694). Padcev (imitation) was included as a comparison. Isotype control compound 25ADC had minimal activity.

FIG. 33 shows a graph of NCI-H1781 xenograft studies on day 0 using single 2.5 or 7.5mg/kg intravenous doses of either the listed anti-nectin-4 or isotype control ADCs. VH4/VL5 compound 25 in DAR4 format (RED-694) using a 91N tag and VH4/VL5 compound 25 in DAR8 format (RED-694. Padcev (mock) using a 91N/116E ditag combination were included as a comparison.

FIG. 34. Double labeled Nectin-4 VH4/VL1 antibody conjugated to Compound 8 produced 3.74 DAR as determined by PLRP.

FIG. 35 double labeled Nectin-4 VH4/VL1 antibody conjugated to Compound 8 was 98.5% monomer as determined by SEC.

FIG. 36A double labeled Nectin-4 VH4/VL5 antibody conjugated to Compound 8 produced a DAR of 3.73 as determined by PLRP.

FIG. 37 double labeled Nectin-4 VH4/VL5 antibody conjugated to Compound 8 was 98.0% monomer as determined by SEC.

FIG. 38 DAR of 6.89 as determined by PLRP was generated by the double labeled Nectin-4 VH4/VL1 antibody conjugated to compound 25.

FIG. 39 double labeled Nectin-4 VH4/VL1 antibody conjugated to compound 25 was 98.7% monomer as determined by SEC.

FIG. 40A double labeled Nectin-4 VH4/VL5 antibody conjugated to compound 25 produced a DAR of 6.86 as determined by PLRP.

FIG. 41 double labeled Nectin-4 VH4/VL5 antibody conjugated to compound 25 was 96.6% monomer as determined by SEC.

FIG. 42. Single labeled Nectin-4 VH4/VL1 antibody conjugated to Compound 25 produced a DAR of 3.16 as determined by PLRP.

FIG. 43 Single labeled Nectin-4 VH4/VL1 antibody conjugated to Compound 25 was 97.2% monomer as determined by SEC.

FIG. 44. Single labeled Nectin-4 VH4/VL5 antibody conjugated to Compound 25 produced DAR of 3.25 as determined by PLRP.

FIG. 45A single labeled Nectin-4 VH4/VL5 antibody conjugated to compound 25 produced a DAR of 3.25 as determined by PLRP.

Fig. 46. Clinical observations of rats repeatedly given cross-reactive nectin-4 ADC. Arrows indicate days of administration. No observation was seen in animals given the compound 25 conjugate, whereas clinical observations in Bei Duoting (vedotin) administration group averaged 2.5 on day 17 and eventually led to death of the animals.

Fig. 47. Erythrocyte counts in rats repeatedly dosed with vehicle or ADC.

Fig. 48 neutrophil counts in rats repeatedly dosed with vehicle or ADC.

Figure 49 reticulocyte counts in rats repeatedly administered vehicle or ADC.

Figure 50 lymphocyte counts in rats repeatedly dosed with vehicle or ADC.

Fig. 51 platelet counts in rats repeatedly administered vehicle or ADC.

Fig. 52 alanine aminotransferase counts in rats repeatedly dosed with vehicle or ADC.

FIG. 53 counts of aspartate aminotransferase in rats repeatedly dosed with vehicle or ADC.

FIG. 54 shows a pharmacokinetic analysis of rat plasma samples from a multi-dose non-GLP rat toxicology study # 2. This analysis confirms the dosage level and shows that the enrolment mab compound 8 conjugate improves in vivo stability over the enrolment mab bepotastine conjugate.

Definition of the definition

"Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, and for example 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. The term includes, for example, straight and branched hydrocarbon groups such as methyl (CH ₃ -), ethyl (CH ₃CH₂ -), n-propyl (CH ₃CH₂CH₂ -), isopropyl ((CH ₃)₂ CH-), n-butyl (CH ₃CH₂CH₂CH₂ -), isobutyl ((CH ₃)₂CHCH₂ -), sec-butyl ((CH ₃)(CH₃CH₂) CH-), tert-butyl ((CH ₃)₃ C-), n-pentyl (CH ₃CH₂CH₂CH₂CH₂ -), and neopentyl ((CH ₃)₃CCH₂ -).

The term "substituted alkyl" refers to an alkyl group as defined herein wherein one or more carbon atoms (other than the C1 carbon atom) in the alkyl chain have optionally been replaced with heteroatoms such as-O-, -N-, -S (O) _n - (wherein N is 0 to 2), -NR- (wherein R is hydrogen or alkyl), and having 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -aryl, SO ₂ -heteroaryl, and-NR ^aR^b, wherein R' and R "may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

"Alkylene" means a divalent aliphatic hydrocarbon group preferably having 1 to 6, and more preferably 1 to 3 carbon atoms, which is straight or branched and optionally interrupted with one or more groups selected from-O-, -NR ¹⁰-、-NR¹⁰C(O)-、-C(O)NR¹⁰ -, and the like. The term includes, for example, methylene (-CH ₂ -), ethylene (-CH ₂CH₂ -), n-propylene (-CH ₂CH₂CH₂ -), isopropylidene (-CH₂CH(CH₃)-)、(-C(CH₃)₂CH₂CH₂-)、(-C(CH₃)₂CH₂C(O)-)、(-C(CH₃)₂CH₂C(O)NH-)、(-CH(CH₃)CH₂-), and the like.

"Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens substituted with substituents described for carbon in the definition of "substituted" below.

The term "alkane" refers to an alkyl group and an alkylene group as defined herein.

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

The term "alkylaryl" or "aralkyl" refers to the groups-alkylene-aryl and substituted alkylene-aryl, wherein alkylene, substituted alkylene, and aryl are as defined herein.

"Alkoxy" refers to the group-O-alkyl, wherein alkyl is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term "substituted alkoxy" refers to the group substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

The term "alkoxyamino" refers to the group-NH-alkoxy, wherein alkoxy is as defined herein.

The term "haloalkoxy" refers to the group alkyl-O-, wherein one or more hydrogen atoms on the alkyl group have been replaced with a halogen group, and includes, for example, groups such as trifluoromethoxy and the like.

The term "haloalkyl" refers to a substituted alkyl group as described above wherein one or more hydrogen atoms on the alkyl group have been replaced with a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

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

The term "alkylthio-alkoxy" refers to the groups-alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl, wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

"Alkenyl" refers to a straight or branched hydrocarbon radical having 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably 1 to 2 sites of double bond unsaturation. The term includes, for example, butadienyl, allyl and but-3-en-1-yl. The term includes both cis and trans isomers or mixtures of these isomers.

The term "substituted alkenyl" refers to an alkenyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, and-SO ₂ -heteroaryl.

"Alkynyl" refers to a straight or branched monovalent hydrocarbon group having 2 to 6 carbon atoms and 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 ethynyl (-c≡ch) and propargyl (-CH ₂ c≡ch).

The term "substituted alkynyl" refers to an alkynyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

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

"Acyl" refers to the groups H-C (O) -, alkyl-C (O) -, substituted alkyl-C (O) -, alkenyl-C (O) -, substituted alkenyl-C (O) -, alkynyl-C (O) -, substituted alkynyl-C (O) -, cycloalkyl-C (O) -, substituted cycloalkyl-C (O) -, and 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) -, 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. For example, acyl groups include the "acetyl" group CH ₃ C (O) -.

"Acylamido" means a radical-NR ²⁰ C (O) alkyl, -NR ²⁰ C (O) substituted alkyl, NR ²⁰ C (O) cycloalkyl, -NR ²⁰ C (O) substituted cycloalkyl-NR ²⁰ C (O) cycloalkenyl, -NR ²⁰ C (O) substituted cycloalkenyl, -NR ²⁰ C (O) alkenyl, -NR ²⁰ C (O) substituted alkenyl-NR ²⁰ C (O) alkynyl, -NR ²⁰ C (O) substituted alkynyl, -NR ²⁰ C (O) aryl, -NR ²⁰ C (O) substituted aryl, -NR ²⁰ C (O) heteroaryl, -NR ²⁰ C (O) substituted heteroaryl, -NR ²⁰ C (O) heterocyclyl, and-NR ²⁰ C (O) substituted heterocyclyl, wherein R ²⁰ is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"Aminocarbonyl" or the term "aminoacyl" refers to the group-C (O) NR ²¹R²², where R ²¹ and R ²² are 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 where R ²¹ and R ²² are optionally linked together with the nitrogen to which they are bonded to form a heterocyclyl or substituted heterocyclyl group, and 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.

"Aminocarbonylamino" refers to the group-NR ²¹C(O)NR²²R²³ wherein R ²¹、R²² and R ²³ are independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or wherein two R groups are joined to form a heterocyclyl group.

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

The term "acyloxy" refers to the groups 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-, wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

"Aminosulfonyl" refers to the group-SO ₂NR²¹R²², wherein R ²¹ and R ²² are 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, substituted heterocyclyl, and wherein R ²¹ and R ²² are optionally linked together with the nitrogen to which they are bound to form a heterocyclyl or substituted heterocyclyl group, and 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.

"Sulfonylamino" means a group-NR ²¹SO₂R²², where R ²¹ and R ²² are 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 where R ²¹ and R ²² are optionally linked together with the atom to which they are bonded to form a heterocyclyl or substituted heterocyclyl group, and 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.

"Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as found in phenyl groups) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracenyl and indanyl), wherein the fused rings may or may not be aromatic rings, provided that the point of attachment is through an atom of an aromatic ring. The term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of aryl substituent, such aryl groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of 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, amido, alkylaryl, aryl, aryloxy, azido, carboxy, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl, and trihalomethyl.

"Aryloxy" refers to a group-O-aryl, wherein aryl is as defined herein, including, for example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups also as defined herein.

"Amino" refers to the group-NH ₂.

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

The term "azido" refers to the group-N ₃.

"Carboxy" (Carboxyl), "carboxy" (carboxy) or "carboxylate" refers to-CO ₂ H or a salt thereof.

"Carboxyester (Carboxyl ester)" or "carboxyester (carboxy ester)" or the term "carboxyalkyl (carboxyalkyl)" or "carboxyalkyl (carboxylalkyl)" refers to the group-C (O) O-alkyl, -C (O) O-substituted alkyl, -C (O) O-alkenyl, -C (O) O-substituted alkenyl, -C (O) O-alkynyl, -C (O) O-substituted alkynyl, -C (O) O-aryl, -C (O) O-substituted aryl, -C (O) O-cycloalkyl, -C (O) O-substituted cycloalkyl, -C (O) O-cycloalkenyl, -C (O) O-substituted cycloalkenyl, -C (O) O-heteroaryl, -C (O) O-substituted heteroaryl, and-C (O) O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted heteroaryl, substituted heteroaryl, heterocyclyl, and the definitions herein.

"(Carboxylester) oxy" or "carbonate" refers to the group-O-C (O) O-alkyl, -O-C (O) O-substituted alkyl, -O-C (O) O-alkenyl-O-C (O) O-substituted alkenyl, -O-C (O) O-alkynyl, -O-C (O) O-substituted alkynyl, -O-C (O) O-aryl, -O-C (O) O-substituted aryl-O-C (O) O-cycloalkyl, -O-C (O) O-substituted cycloalkyl, -O-C (O) O-cycloalkenyl, -O-C (O) O-substituted cycloalkenyl, -O-C (O) O-heteroaryl, -O-C (O) O-substituted heteroaryl, -O-C (O) O-heterocyclyl, and-O-C (O) O-substituted heterocyclyl, 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.

"Cyano" or "nitrile" refers to the group-CN.

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

The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

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

The term "substituted cycloalkenyl" refers to cycloalkenyl groups having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, and-SO ₂ -heteroaryl.

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

"Cycloalkoxy" refers to-O-cycloalkyl.

"Cycloalkenyloxy" refers to an-O-cycloalkenyl group.

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

"Hydroxy" refers to the group-OH.

"Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms in the ring, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups may have a single ring (such as pyridyl, imidazolyl or furyl) or multiple condensed rings in a ring system (e.g., in a group such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To meet valence requirements, any heteroatom in such heteroaryl ring may or may not be bonded to H or a substituent, such as an alkyl group or other substituent described herein. In certain embodiments, the nitrogen and/or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide an N-oxide (n→o), sulfinyl, or sulfonyl moiety. The term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl and furyl. Unless otherwise limited by the definition of heteroaryl substituent, such heteroaryl groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from the group consisting of 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, amido, alkylaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl, and trihalomethyl.

The term "heteroarylalkyl" refers to the group-alkylene-heteroaryl, wherein alkylene and heteroaryl are as defined herein. The term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

"Heteroaryloxy" refers to an-O-heteroaryl group.

"Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings (including fused bridged and spiro ring systems) and having from 3 to 20 ring atoms (including 1 to 10 heteroatoms). These ring atoms are selected from nitrogen, sulfur or oxygen, wherein in the fused ring system one or more of the rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S (O) -or-SO ₂ -moiety. To meet valence requirements, any heteroatom in such heterocycle may or may not be bonded to one or more H or one or more substituents, such as alkyl groups or other substituents described herein.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidines, pyrroles, imidazoles, pyrazoles, pyridines, pyrazines, pyrimidines, pyridazines, indolines, indoles, indolines, indazoles, purines, quinolizines, isoquinolines, quinolines, phthalazines, naphthyridines, quinoxalines, quinazolines, cinnolines, pteridines, carbazoles, carbolines, phenanthridines, acridines, phenanthrolines, isothiazoles, phenazines, isoxazoles, phenoxazines, phenothiazines, imidazolidines, imidazolines, piperidines, piperazines, indolines, phthalimides, 1,2,3, 4-tetrahydroisoquinolines, 4,5,6, 7-tetrahydrobenzo [ b ] thiophenes, thiazoles, thiazolidines, thiophenes, benzo [ b ] thiophenes, morpholines, thiomorpholines (also known as thiomorpholines), 1-dioxothiomorpholines, piperidines, pyrrolidines, tetrahydrofurans, and the like.

Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl and fused heterocycles.

"Heterocyclyloxy" refers to the group-O-heterocyclyl.

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

The term "heterocycle" refers to a diradical group formed from a heterocycle as defined herein.

The term "hydroxyamino" refers to the group-NHOH.

"Nitro" refers to the group-NO ₂.

"Oxo" refers to an atom (=o).

"Sulfonyl" refers to the groups SO ₂ -alkyl, SO ₂ -substituted alkyl, SO ₂ -alkenyl, SO ₂ -substituted alkenyl, SO ₂ -cycloalkyl, SO ₂ -substituted cycloalkyl, SO ₂ -cycloalkenyl, SO ₂ -substituted cycloalkenyl, SO ₂ -aryl, SO ₂ -substituted aryl, SO ₂ -heteroaryl, SO ₂ -substituted heteroaryl, SO ₂ -heterocyclyl and SO ₂ -substituted heterocyclyl, 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. Sulfonyl groups include, for example, methyl-SO ₂ -, phenyl-SO ₂ -, and 4-methylphenyl-SO ₂ -.

"Sulfonyloxy" refers to the groups-OSO ₂ -alkyl, OSO ₂ -substituted alkyl, OSO ₂ -alkenyl, OSO ₂ -substituted alkenyl, OSO ₂ -cycloalkyl, OSO ₂ -substituted cycloalkyl, OSO ₂ -cycloalkenyl, OSO ₂ -substituted cycloalkenyl, OSO ₂ -aryl, OSO ₂ -substituted aryl, OSO ₂ -heteroaryl, OSO ₂ -substituted heteroaryl, OSO ₂ -heterocyclyl, and OSO ₂ -substituted heterocyclyl, 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.

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

"Thiol" refers to the group-SH.

"Thio (keto) group" or the term "thioketo" refers to an atom (=s).

"Alkylthio" or the term "thioalkoxy" refers to the group-S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to-S (O) -. Sulfoxides may exist as one or more stereoisomers.

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

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

The term "thioheteroaryloxy" refers to the group heteroaryl-S-, wherein heteroaryl groups are as defined herein, including optionally substituted aryl groups also as defined herein.

The term "thioheterocyclyloxy" refers to the group heterocyclyl-S-, wherein heterocyclyl groups are as defined herein, including optionally substituted heterocyclyl groups also as defined herein.

In addition to the disclosure herein, the term "substituted" when used to modify a particular group or radical may also refer to one or more hydrogen atoms of the particular group or radical each being substituted, independently of the other, by the same or different substituents as defined below.

In addition to the groups disclosed with respect to the various terms herein, unless otherwise indicated, the substituents for substitution of one OR more hydrogens on saturated carbon atoms in a particular group OR radical (any two hydrogens on a single carbon may be replaced with =o, =nr ⁷⁰、＝N-OR⁷⁰、＝N₂ OR =s) are-R ⁶⁰, halogen, =o, -OR ⁷⁰、-SR⁷⁰、-NR⁸⁰R⁸⁰, trihalomethyl 、-CN、-OCN、-SCN、-NO、-NO₂、＝N₂、-N₃、-SO₂R⁷⁰、-SO₂O^–M⁺、-SO₂OR⁷⁰、-OSO₂R⁷⁰、-OSO₂O^–M⁺、-OSO₂OR⁷⁰、-P(O)(O^–)₂(M⁺)₂、-P(O)(OR⁷⁰)O^–M⁺、-P(O)(OR⁷⁰)₂、-C(O)R⁷⁰、-C(S)R⁷⁰、-C(NR⁷⁰)R⁷⁰、-C(O)O^–M⁺、-C(O)OR⁷⁰、-C(S)OR⁷⁰、-C(O)NR⁸⁰R⁸⁰、-C(NR⁷⁰)NR⁸⁰R⁸⁰、-OC(O)R⁷⁰、-OC(S)R⁷⁰、-OC(O)O^-M⁺、-OC(O)OR⁷⁰、-OC(S)OR⁷⁰、-NR⁷⁰C(O)R⁷⁰、-NR⁷⁰C(S)R⁷⁰、-NR⁷⁰CO₂ ^–M⁺、-NR⁷⁰CO₂R⁷⁰、-NR⁷⁰C(S)OR⁷⁰、-NR⁷⁰C(O)NR⁸⁰R⁸⁰、-NR⁷⁰C(NR⁷⁰)R⁷⁰ and-NR ⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R ⁶⁰ is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R ⁷⁰ is independently hydrogen OR R ⁶⁰; each R ⁸⁰ is independently R ⁷⁰ or alternatively two R ^80' together with the nitrogen atom to which they are bound form a 5-, 6-or 7-membered heterocycloalkyl, which may optionally include 1 to 4 identical or different further heteroatoms selected from the group consisting of O, N and S, wherein N may have-H or C ₁-C₃ alkyl substitution; and each M ⁺ is a counterion with a net single positive charge. Each M ⁺ can independently be, for example, a base ion, such as K ⁺、Na⁺、Li⁺; ammonium ions, such as ⁺N(R⁶⁰)₄; or alkaline earth ions such as [ Ca ²⁺]_0.5、[Mg²⁺]_0.5 or [ Ba ²⁺]_0.5 (subscript 0.5 means that one of the counter ions of a divalent alkaline earth ion may be an ionized form of a compound of the invention, and another typical counter ion such as chloride, or two ionizing compounds disclosed herein may act as counter ions for such divalent alkaline earth ions, or a dual ionizing compound of the invention may act as counter ions for such divalent alkaline earth ions). As specific examples, -NR ⁸⁰R⁸⁰ is meant to include-NH ₂, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

In addition to the disclosure herein, unless otherwise indicated, substituents for hydrogen on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are-R ⁶⁰, halogen, -O ^-M⁺、-OR⁷⁰、-SR⁷⁰、-S^–M⁺、-NR⁸⁰R⁸⁰, trihalomethyl 、-CF₃、-CN、-OCN、-SCN、-NO、-NO₂、-N₃、-SO₂R⁷⁰、-SO₃ ^–M⁺、-SO₃R⁷⁰、-OSO₂R⁷⁰、-OSO₃ ^–M⁺、-OSO₃R⁷⁰、-PO₃ ^-2(M⁺)₂、-P(O)(OR⁷⁰)O^–M⁺、-P(O)(OR⁷⁰)₂、-C(O)R⁷⁰、-C(S)R⁷⁰、-C(NR⁷⁰)R⁷⁰、-CO₂ ^–M⁺、-CO₂R⁷⁰、-C(S)OR⁷⁰、-C(O)NR⁸⁰R⁸⁰、-C(NR⁷⁰)NR⁸⁰R⁸⁰、-OC(O)R⁷⁰、-O C(S)R⁷⁰、-OCO₂ ^–M⁺、-OCO₂R⁷⁰、-OC(S)OR⁷⁰、-NR⁷⁰C(O)R⁷⁰、-NR⁷⁰C(S)R⁷⁰、-NR⁷⁰CO₂ ^–M⁺、-NR⁷⁰CO₂R⁷⁰、-NR⁷⁰C(S)OR⁷⁰、-NR⁷⁰C(O)NR⁸⁰R⁸⁰、-NR⁷⁰C(NR⁷⁰)R⁷⁰, and-NR ⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R ⁶⁰、R⁷⁰、R⁸⁰ and M ⁺ are as previously defined, provided that in the case of a substituted alkene or alkyne, the substituents are not-O ^-M⁺、-OR⁷⁰、-SR⁷⁰ or-S ^–M⁺.

Except as disclosed with respect to the various terms herein, unless otherwise indicated, the substituents for hydrogen on the nitrogen atom in the "substituted" heteroalkyl and cycloheteroalkyl groups are -R⁶⁰、-O^-M⁺、-OR⁷⁰、-SR⁷⁰、-S^-M⁺、-NR⁸⁰R⁸⁰、 trihalomethyl 、-CF₃、-CN、-NO、-NO₂、-S(O)₂R⁷⁰、-S(O)₂O^-M⁺、-S(O)₂OR⁷⁰、-OS(O)₂R⁷⁰、-OS(O)₂O^-M⁺、-OS(O)₂OR⁷⁰、-P(O)(O^-)₂(M⁺)₂、-P(O)(OR⁷⁰)O^-M⁺、-P(O)(OR⁷⁰)(OR⁷⁰)、-C(O)R⁷⁰、-C(S)R⁷⁰、-C(NR⁷⁰)R⁷⁰、-C(O)OR⁷⁰、-C(S)OR⁷⁰、-C(O)NR⁸⁰R⁸⁰、-C(NR⁷⁰)NR⁸⁰R⁸⁰、-OC(O)R⁷⁰、-OC(S)R⁷⁰、-OC(O)OR⁷⁰、-OC(S)OR⁷⁰、-NR⁷⁰C(O)R⁷⁰、-NR⁷⁰C(S)R⁷⁰、-NR⁷⁰C(O)OR⁷⁰、-NR⁷⁰C(S)OR⁷⁰、-NR⁷0C(O)NR⁸⁰R⁸⁰、-NR⁷⁰C(NR⁷⁰)R⁷⁰ and-NR ⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R ⁶⁰、R⁷⁰、R⁸⁰ and M ⁺ are as previously defined.

In addition 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.

It is to be understood that among all the substituent groups defined above, polymers obtained by limiting substituents having additional substituents to themselves (e.g., substituted aryl groups having substituted aryl groups as substituents themselves substituted with substituted aryl groups (which are further substituted with substituted aryl groups, etc.) are not intended to be included herein.

Unless otherwise indicated, the naming of substituents not explicitly defined herein is derived by naming the terminal portion of a functional group followed by an adjacent functional group towards the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl) - (alkyl) -O-C (O) -.

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

The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient (such as a mammal) (a salt having a counterion that is acceptable for mammalian safety for a given dosage regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of the compounds which are derived from a variety of organic and inorganic counterions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains basic functional groups, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

The term "salt thereof" refers to a compound formed when the protons of an acid are replaced with cations such as metal cations or organic cations. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended for administration to a patient. For example, salts of the compounds of the invention include salts in which the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

"Solvate" refers to a complex formed by the binding of a solvent molecule to a molecule or ion of a solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

"Stereoisomer (Stereoisomer)" refers to a compound having the same atom connectivity but different in the spatial arrangement of atoms. Stereoisomers include cis, trans, E and Z isomers, enantiomers and diastereomers.

"Tautomer" refers to alternative forms of molecules with only electron bonding of atoms and/or position difference of protons, such as enol-ketone and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing an array of-n=c (H) -NH-ring atoms, 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.

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

The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., igG1, igG2, igG3, or IgG 4), igE, igD, igA, igM, etc.); whole antibodies (e.g., antibodies comprising tetramers, which in turn comprise two dimers of heavy and light chain polypeptides); single chain antibodies (e.g., scFv); antibody fragments (e.g., fragments of whole chain or single chain antibodies) that retain specific binding to an antigen, including but not limited to Fab, fv, scFv and Fd fragments; a chimeric antibody; a humanized antibody; a single chain antibody and a fusion protein comprising an antigen binding portion of the antibody and a non-antibody protein. The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or the like. The antibodies may be further conjugated to other moieties such as members of specific binding pairs, e.g., biotin (a member of a biotin-avidin specific binding pair), and the like. Antibodies may also be bound to solid supports including, but not limited to, polystyrene plates or beads, and the like. The term also includes Fab ', fv, F (ab') ₂ and or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. Antibodies may be monovalent or bivalent.

An "antibody fragment" includes a portion of an intact antibody, such as an antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') ₂, and Fv fragments; a diabody; linear antibodies (Zapata et al, protein Eng.8 (10): 1057-1062 (1995)); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. Pepsin treatment resulted in a F (ab') ₂ fragment with two antigen binding sites and still capable of cross-linking the antigen.

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

The "Fab" fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH ₁). Fab fragments are distinguished from Fab' fragments by the addition of several residues (including one or more cysteines from the antibody hinge region) at the carboxy terminus of the heavy chain CH ₁ domain. Fab '-SH is the designation herein for Fab' in which the cysteine residue of the constant domain bears a free thiol group. The F (ab ') ₂ antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinct types (termed kappa and lambda) based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the constant domains of their heavy chains. There are five main classes of immunoglobulins: igA, igD, igE, igG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA and IgA2.

"Single chain Fv" or "sFv" antibody fragments include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some aspects, fv polypeptides further comprise a polypeptide linker (linker) between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.

The term "diabody" refers to a small antibody fragment having two antigen binding sites, the fragment comprising a heavy chain variable domain (V _H) linked to a light chain variable domain (V _L) in the same polypeptide chain (V _H-V_L). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and create two antigen binding sites.

As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents and is expressed as the dissociation constant (Kd). The affinity may 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 than the affinity of the antibody for the unrelated amino acid sequence. The affinity of the antibody for the target protein may be, for example, from about 100 nanomolar (nM) to about 0.1nM, from about 100nM to about 1 picomolar (pM), or from about 100nM to about 1 femtomole (fM) or more. As used herein, the term "affinity (avidity)" refers to the resistance of a complex of two or more agents to dissociation after dilution. With respect to antibodies and/or antigen binding fragments, the terms "immunocompetent" and "preferentially binding" are used interchangeably herein.

The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen bond interactions (including interactions such as salt and water bridges). The subject anti-Nectin-4 antibody specifically binds to an epitope within a Nectin-4 polypeptide (e.g., a human Nectin-4 polypeptide, such as glycosylated Nectin-4 or a fragment thereof). Non-specific binding will refer to binding with an affinity of less than about 10 ^-7 M, such as binding with an affinity of 10 ^-6M、10^-5M、10^-4 M, etc.

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

"High affinity" binding refers to binding having a K _a of at least 10 ⁷M^-1, at least 10 ⁸M^-1, at least 10 ⁹M^-1, at least 10 ¹⁰M^-1, at least 10 ¹¹M^-1, at least 10 ¹²M^-1, at least 10 ¹³M^-1, or greater. Alternatively, affinity may be defined as the equilibrium dissociation constant (K _D) of a particular binding interaction in units of M (e.g., 10 ^-5 M to 10 ^-13 M, or less). In some embodiments, specific binding means that the antibody binds to the antigen with a K _D of less than or equal to about 10 ^-5 M, less than or equal to about 10 ^-6 M, less than or equal to about 10 ^-7 M, less than or equal to about 10 ^-8 M, or less than or equal to about 10 ^-9M、10^-10M、10^-11 M, or 10 ^-12 M or less. The binding affinity of an antibody for an antigen can be readily determined using conventional techniques, e.g., by competition ELISA (enzyme linked immunosorbent assay), equilibrium dialysis, by using Surface Plasmon Resonance (SPR) techniques (e.g., BIAcore 2000 instruments, using general procedures outlined by the manufacturer), by radioimmunoassay, and the like.

As used herein, the term "CDR" or "complementarity determining region" means a discontinuous antigen binding site found within the variable regions of both heavy and light chain polypeptides. CDRs have been described by Kabat et al, J.biol. Chem.252:6609-6616 (1977); kabat et al, dept.of HEALTH AND Human Services, "Sequences of proteins of immunological interest" (1991); chothia et al, J.mol.biol.196:901-917 (1987); and MacCallum et al, J.mol.biol.262:732-745 (1996), wherein the definition includes overlapping or subsets of amino acid residues when compared to each other. However, any definition is applied to refer to CDRs of an antibody or grafted antibody or variant thereof, and is intended to be within the scope of the terms as defined and used herein. By way of comparison, amino acid residues encompassing CDRs as defined by each of the references cited above are set forth in table 1 below.

TABLE 1 CDR definition

	Kabat¹	Chothia²	MacCallum³
				V_H CDR1	31-35	26-32	30-35
V_H CDR2	50-65	53-55	47-58
				V_H CDR3	95-102	96-101	93-101
V_L CDR1	24-34	26-32	30-36
				V_L CDR2	50-56	50-52	46-55
V_L CDR3	89-97	91-96	89-96

¹ Residue numbering follows the nomenclature of Kabat et al, supra

² Residue numbering follows the nomenclature of Chothia et al described above

³ Residue numbering follows the nomenclature of MacCallum et al above

Throughout this disclosure, the residue numbers in the immunoglobulin heavy and light chains are MacCallum et al, sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991), which is expressly incorporated herein by reference.

As used herein, the term "framework" when used in reference to an antibody variable region means all amino acid residues outside of the CDR regions within the variable region of the antibody. The variable region framework is typically a discontinuous amino acid sequence of between about 100-120 amino acids in length, but means only those amino acids outside of the CDR. As used herein, the term "framework region" means each domain of a framework separated by CDRs.

A "native (native) Ig polypeptide" is a polypeptide comprising an amino acid sequence that lacks an aldehyde-tagged constant region as described herein. The 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).

In the context of Ig polypeptides, the term "constant region" is well known in the art and refers to the C-terminal region of an Ig heavy chain or Ig light chain. Ig heavy chain constant regions include CH1, CH2, and CH3 domains (as well as CH4 domains, wherein the heavy chain is a μ or ε heavy chain). In a natural Ig heavy chain, the CH1, CH2, CH3 (and CH4 if present) domains begin immediately after (the C-terminus of) the heavy chain Variable (VH) region, and each is from about 100 amino acids to about 130 amino acids in length. In a natural Ig light chain, the constant region starts immediately after (the C-terminal end of) the light chain variable region (VL) and is about 100 amino acids to 120 amino acids in length.

An "epitope" is a site on an antigen to which an antibody binds (e.g., a site on Nectin-4). Epitopes can be formed by folding (e.g., tertiary folding) of proteins from either contiguous amino acids in juxtaposition or non-contiguous amino acids. Epitopes formed by consecutive 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 comprise at least 3, and more typically at least 5 or 8-10 amino acids in a linear or spatial conformation. Methods of determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, epitope Mapping Protocols in Methods in Molecular Biology, volume 66, glenn E.Morris edit (1996). Some commercial laboratories offer epitope mapping services. Epitopes bound by antibodies that immunoreact with antigens associated with the membrane may be located on the cell surface (e.g., in the extracellular region of the transmembrane protein), and thus such epitopes are considered to be cell surface accessible, solvent accessible, and/or cell surface exposed.

"Gene-encodable" as used in reference to an amino acid sequence of a polypeptide, peptide or protein means that the amino acid sequence includes amino acid residues that can be produced by transcription and translation of the 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.

The term "control sequence" refers to a DNA sequence that facilitates expression of an operably linked coding sequence in a particular expression system (e.g., mammalian cells, bacterial cells, cell-free synthesis, etc.). Control sequences suitable for use in prokaryotic systems include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA of the presequence or secretory main conductor (secretory leader) is expressed as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate initiation of translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of secretion of the primary conductor, contiguous and in reading frame. Ligation is accomplished by ligation or by an amplification reaction. Synthetic oligonucleotide adaptors (adaptors) or linkers may be used to ligate sequences according to conventional practice.

The term "expression cassette" as used herein refers to a fragment of nucleic acid, typically DNA, that can be inserted into a nucleic acid (e.g., by using restriction sites compatible with conjugation to a construct of interest or by homologous recombination into the construct of interest or the host cell genome). In general, nucleic acid fragments include polynucleotides encoding a polypeptide of interest, and cassettes and restriction sites are designed to facilitate insertion of the cassettes into the correct reading frame for transcription and translation. The expression cassette may also include elements that promote expression of a polynucleotide encoding a polypeptide of interest in a host cell (e.g., a mammalian host cell). These elements may include, but are not limited to: promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

An "isolated" antibody is one that has been identified and isolated and/or recovered from components of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 90%, greater than 95% or greater than 98% by weight, e.g., greater than 99% by weight, of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain at least 15N-terminal residues or internal amino acid sequences by using a rotary cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using coomassie brilliant blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the natural environment of the antibody will not be present. In some cases, the isolated antibody will be prepared by at least one purification step.

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

The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody that contains one or more amino acids (e.g., in the framework regions, constant regions, or CDRs) substituted with amino acids from the corresponding positions of a human antibody. In general, humanized antibodies produce reduced immune responses in a human host compared to non-humanized versions of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting, veneering or surface remodeling, chain substitution (chain grafting), and the like. In certain embodiments, the framework substitutions are identified by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding and sequence alignment to identify aberrant framework residues at specific positions. Thus, the antibodies described above may be humanized using methods well known in the art.

In certain embodiments, the antibody molecules disclosed herein include a polypeptide comprising a variable heavy chain region as provided herein and having a UniProt: heavy chain of human IgG1 constant region of amino acid sequence set forth in P01857-1 version 1. In certain embodiments, an antibody molecule disclosed herein comprises a light chain comprising a variable light chain region as provided herein and a human light chain constant region. In certain embodiments, the human light chain constant region is one having UniProtKB/Swiss-Prot: human kappa light chain constant region of amino acids set forth in P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibody can include mutations, e.g., substitutions that modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A and P329G) or N297A mutations can 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 substitutions is based on the EU numbering system. When residues in the immunoglobulin heavy chain constant region are involved, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991)). "EU index as in Kabat" refers to the residue numbering of the human IgG 1EU antibody.

The term "chimeric antibody" refers to an antibody whose light and heavy chain genes are typically constructed by genetic engineering of antibody variable and constant region genes belonging to different species. For example, variable fragments of genes from mouse monoclonal antibodies may be linked to human constant fragments, such as γ1 and γ3. Examples of therapeutic chimeric antibodies are hybrid proteins comprising a variable or antigen binding domain from a mouse antibody and a constant or effector domain from a human antibody, although domains from other mammalian species may be used.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless explicitly stated otherwise, "polypeptide," "peptide" and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone (backbone). The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., facilitating production in recombinant host cells), immunolabeled proteins, and the like. In the context of antibodies, it is apparent that chains or domains include polypeptides.

"Native amino acid sequence" or "parent amino acid sequence" is used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include modified amino acid residues.

The terms "amino acid analog," "unnatural amino acid," and the like, are used interchangeably and include amino acid-like compounds that are similar in structure and/or overall shape to one or more amino acids typically found in naturally occurring proteins (e.g., ala or A, cys or C, asp or D, glu or E, phe or F, gly or G, his or H, ile or I, lys or K, leu or L, met or M, asn or N, pro or P, gln or Q, arg or R, ser or S, thr or T, val or V, trp or W, tyr or Y). Amino acid analogs also include natural amino acids having modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as in the naturally occurring D-form as well as in the L-form of the amino acid analog. In some cases, amino acid analogs share one or more backbone structures and/or side chain structures of the natural amino acid, with the difference being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond to double bond, etc.), or combinations thereof. For example, amino acid analogs can include alpha-hydroxy acids, alpha-amino acids, and the like.

The term "amino acid side chain" or "side chain of an amino acid" and the like may be used to refer to substituents attached to the alpha-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. The 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.

The term "conjugation" generally refers to chemical attachment (either covalent or non-covalent, typically covalent) that associates one molecule of interest with the proximal end of a second molecule of interest. In some embodiments, the agent is selected from the group consisting of a half-life extending moiety, a labeling agent, and a therapeutic agent. For half-life extension, for example, the antibodies of the disclosure can optionally be modified to provide improved pharmacokinetic profiles (e.g., by pegylation, hyperglycosylation, etc.). Modifications that can enhance serum half-life are of interest.

The term "carbohydrate" and the like may be used to refer to monomers and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates such as monosaccharides, disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (with any convenient substituent), modified (converted to another group using any convenient chemical reaction), or absent (e.g., eliminated or substituted with H). A variety of carbohydrates and carbohydrate derivatives are available and may be suitable for use with the subject compounds and conjugates.

As used herein, the term "isolated" is intended to describe a compound of interest in an environment different from the environment in which the compound naturally occurs. "isolating" is intended to include compounds within a sample wherein the compound of interest is substantially enriched and/or wherein the compound of interest is partially or substantially purified.

As used herein, the term "substantially purified" refers to a compound that is removed from its natural environment and that 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 of other components with which it is naturally associated.

The term "physiological conditions" is intended to encompass those conditions that are compatible with living cells, such as temperature, pH, salinity, etc., that are primarily aqueous.

"Reactive partner" refers to a molecule or portion of a molecule that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include cysteine or serine of the sulfatase motif and Formylglycine Generating Enzymes (FGEs) that react to form a reaction product containing a converted aldehyde tag of formylglycine (fGly) in place of the cysteine or serine in the motif. Other exemplary reactive partners include aldehydes (e.g., reactive aldehyde groups) of the fGly residues of the converted aldehyde tag and "aldehyde-reactive partners" that comprise an aldehyde-reactive group and a moiety of interest and which react to form a reaction product of a polypeptide having a moiety of interest conjugated to the polypeptide through fGly residues.

"N-terminal" refers to the terminal amino acid residue of a polypeptide having a free amine group, the amine group in a non-N-terminal amino acid residue typically forming part of the covalent backbone of the polypeptide.

"C-terminal" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, the carboxyl group in a non-C-terminal amino acid residue typically forming part of the covalent backbone of the polypeptide.

"Internal site" as used when referring to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or C-terminus.

As used herein, the term "treatment" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effects attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (particularly in a human) and includes: (a) Preventing a disease from occurring in a subject who is likely to be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, e.g., causing regression of the disease.

The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to a mammal, including but not limited to, a mouse (rat, mouse), a non-human primate, a human, a canine, a feline, an ungulate (e.g., equine, bovine, ovine, porcine, caprine), and the like.

"Therapeutically effective amount" or "effective amount" refers to an amount of a subject anti-Nectin-4 antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment of the disease. The "therapeutically effective amount" will vary depending on the anti-Nectin-4 antibody, the disease and its severity, the age, weight, etc., of the subject to be treated.

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

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

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

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

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

Detailed Description

The present disclosure provides antibodies specific for Nectin-4. The disclosure also provides antibody conjugates, such as ADCs, comprising such antibodies specific for Nectin-4. The present disclosure provides methods of producing such antibodies and conjugates, and methods of using the same. Each of the embodiments is described in more detail in the following sections. Also provided are compositions, including in some cases pharmaceutical compositions, comprising the antibodies and/or ADCs of the present disclosure. In certain aspects, methods of using an ADC are provided that include administering a therapeutically effective amount of an ADC of the present disclosure to an individual having a cell proliferative disorder.

NECTIN-4 antibodies and antibody-drug conjugates thereof

As summarized above, the present disclosure provides antibodies specific for Nectin-4 and conjugates of such antibodies (e.g., antibody-drug-conjugates (ADCs)). In addition, the present disclosure provides anti-Nectin-4 antibodies comprising fGly residues.

Antibody-drug conjugates

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

In certain embodiments, the conjugate is a polypeptide conjugate that includes a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, the one or more moieties conjugated to the polypeptide may each independently be any of a variety of moieties of interest, such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilizing the polypeptide to a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, wherein the polypeptide is an antibody, thereby providing an antibody-drug conjugate. For example, the conjugate may 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 conjugates and are described in more detail below.

The one or more drugs or active agents may be conjugated to the polypeptide (e.g., antibody) at any desired site of the polypeptide. Thus, the present disclosure provides polypeptides having conjugated one or more drugs or active agents, for example, at a site at or near the C-terminus of the polypeptide. Other examples include polypeptides having one or more drugs or active agents conjugated at a position at or near the N-terminus of the polypeptide. Examples also include polypeptides having one or more drugs or active agents conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible when the polypeptide is conjugated to more than one drug or active agent.

In certain embodiments, the conjugates of the present disclosure include one or more drugs or active agents conjugated to an amino acid residue of a polypeptide at the α -carbon of the amino acid residue. In other words, conjugates include polypeptides in which the side chains of one or more amino acid residues in the polypeptide have been modified and attached to one or more drugs or active agents (e.g., through a linker as described herein). For example, conjugates include polypeptides in which the alpha-carbon of one or more amino acid residues in the polypeptide has been modified and attached to one or more drugs or active agents (e.g., via a linker as described herein).

Embodiments of the present disclosure include conjugates in which the polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 moieties or more. The moiety may be conjugated to the polypeptide at one or more sites of the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of a polypeptide. In some cases, one moiety is conjugated to an amino acid residue of a polypeptide. In other embodiments, both moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, the first moiety is conjugated to a first amino acid residue of the polypeptide and the second moiety is conjugated to a second amino acid residue of the polypeptide. In other embodiments, both moieties may be conjugated to a first amino acid residue of a polypeptide and both moieties may be conjugated to a second amino acid residue of a polypeptide. Combinations of the above are also possible, for example wherein the polypeptide is conjugated to a first moiety at a first amino acid residue and to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to the first and second portions at a first amino acid residue and conjugated to the third and fourth portions at a second amino acid residue, and the like.

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

In certain embodiments, the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) are conjugated via a conjugate moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bound) to the conjugate moiety, such that the polypeptide and the moiety of interest are indirectly bound by the conjugate moiety. In some cases, the conjugate moiety comprises a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound, or a derivative of a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound. For example, the general scheme for coupling a moiety of interest to a polypeptide via a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety is shown in the general reaction scheme below. The hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl conjugate moieties are also referred to herein as hydrazino-iso-Pictet-Spengler (HIPS) conjugate moiety and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugate moiety, respectively.

In the above reaction schemes, 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), where n is 0 or an integer from 1 to 4. As shown in the above reaction schemes, a conjugate moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety) is attached to one or more drugs or active agents R. A polypeptide (e.g., an antibody) comprising a 2-formylglycine residue (fGly) is reacted with a conjugate moiety to produce a polypeptide (e.g., an antibody) conjugate, thereby attaching one or more drugs or active agents to the polypeptide through the conjugate moiety.

As described herein, the moiety of interest (also referred to herein as a "payload") may be any of a variety of moieties, such as, but not limited to, a chemical entity, such as a detectable label, or a drug or active agent. R 'and R' may each independently be any desired substituent such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z may be CR ²¹、NR²², N, O, or S, wherein R ²¹ and R ²² are each independently selected from any of the substituents described above for R 'and R'.

Other hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties are also possible, as shown in the conjugates and compounds described herein. For example, a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety may be attached (e.g., covalently attached) to one or more linkers. Thus, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties attached to one or more drugs or active agents via corresponding linkers. Thus, conjugates of the present disclosure may include one or more linkers, wherein each linker attaches one or more corresponding drugs or active agents to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. In some cases, a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety and one or more linkers may be generally considered "branched linkers" in which the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety is attached to two or more "branches" in which each branch comprises a linker attached to a drug or active agent.

For example, in some cases of the above reaction schemes, n is 0, and thus one R group (e.g., a drug or active agent) is attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety through a linker. In other cases, n is 1, and thus two R groups (e.g., a drug or active agent) are each attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety via their own respective linkers. In these cases, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety and the two linkers may be generally considered branched linkers.

Combinations of the same or different loads may be conjugated to the polypeptide via branched linkers. In certain embodiments, the two loads (e.g., drug, active agent, or detectable label) attached to the branched linker are the same load (e.g., drug, active agent, or detectable label). For example, a first branch of a branched linker may be attached to a load (e.g., a drug, an active agent, or a detectable label), and a second branch of a branched linker may be attached to the same load as the first branch (e.g., a drug, an active agent, or a detectable label).

In other embodiments, the two loads (e.g., drug, active agent, or detectable label) attached to the branched linker are different loads (e.g., drug, active agent, or detectable label). For example, a first branch of a branched linker may be attached to a first load (e.g., a first drug, active agent, or detectable label), and a second branch of the branched linker may be attached to a second load (e.g., a second drug, active agent, or detectable label) that is different from the first load (e.g., the first drug, active agent, or detectable label) attached to the first branch.

Various embodiments of linkers that can couple a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate 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 the cleavable linkers described herein.

In certain embodiments, the polypeptide may be conjugated to one or more moieties of interest, wherein one or more amino acids of the polypeptide are modified prior to conjugation to the one or more moieties of interest. Modification of one or more amino acids of the polypeptide may result in a polypeptide containing one or more reactive groups suitable for conjugation to one or more moieties of interest. In some cases, the polypeptide may include one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to one or more moieties of interest. For example, an amino acid of a polypeptide can be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). The reactive aldehyde may be included in an "aldehyde tag" or "aldehyde-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 herein as "fGly") by the action of a Formylglycine Generating Enzyme (FGE). The fGly residue produced by FGE may also be referred to as "formylglycine". In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence that includes a "converted" sulfatase motif, i.e., a sulfatase motif in which a cysteine or serine residue has been converted to fGly by the action of FGE, e.g., L (fGly) TPSR (SEQ ID NO: 245). The converted sulfatase motif may be produced from an amino acid sequence comprising an "unconverted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has not been converted to fGly by FGE but which is capable of being converted, e.g., an unconverted sulfatase motif having the sequence L (C/S) TPSR). "transformation" as used in the context of the action of Formylglycine Generating Enzyme (FGE) on a sulfatase motif refers to the biochemical modification of a cysteine or serine residue in the sulfatase motif to a formylglycine (fGly) residue (e.g., cys to fGly, or Ser to fGly). Other aspects of aldehyde tags and their use in site-specific protein modification are described in U.S. patent No. 7,985,783 and U.S. patent No. 8,729,232 (the disclosures of each of which are incorporated herein by reference).

In some cases, to produce a conjugate, a polypeptide containing fGly residues can be conjugated to one or more moieties of interest by reaction of fGly with a compound (e.g., a compound containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, as described above). For example, a polypeptide containing fGly may be contacted with a reactive partner under conditions suitable to provide conjugation of one or more drugs or active agents to the polypeptide. In some cases, the reactive partner may include a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above. For example, one or more drugs or active agents may be attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. In some cases, one or more drugs or active agents are attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, such as covalently attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl, wherein each drug or active agent is attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety via a corresponding linker.

In certain embodiments, conjugates of the present disclosure include polypeptides (e.g., antibodies) 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 prepare the conjugate, the amino acid residues of the polypeptide may be modified and then coupled to one or more drugs or active agents attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above. In certain embodiments, the amino acid residue of a polypeptide (e.g., an antibody) is a cysteine or serine residue that is converted to fGly residues, as described above. In certain embodiments, the converted amino acid residues (e.g., fGly residues) are conjugated to one or more drugs or active agents containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above to provide the conjugates of the present disclosure, wherein the one or more drugs or active agents are conjugated to the polypeptide through the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. As used herein, the term fGly' refers to an amino acid residue of a polypeptide (e.g., an antibody) that is coupled to one or more moieties of interest (e.g., one or more drugs or active agents).

In certain embodiments, conjugates include polypeptides (e.g., antibodies) having at least one amino acid residue attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety described herein, which in turn is attached to one or more drugs or active agents via one or more corresponding linkers. For example, a conjugate can include 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.

Aspects of the disclosure include conjugates of formula (I):

wherein:

Z ¹、Z²、Z³ and Z ⁴ are each independently selected from CR ⁴, N and C-L ^B-W²;

R ¹ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R ² and R ³ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, 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 linked in a cyclic manner to form a 5 or 6 membered heterocyclyl;

Each R ⁴ is independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

l ^A is a first linker;

L ^B is a second linker;

W ¹ is a first drug;

w ² is a second drug; and

W ³ is a polypeptide.

Substituents associated with the conjugates of formula (I) are described in more detail below.

In certain embodiments, Z ¹、Z²、Z³ and Z ⁴ are each independently selected from CR ⁴, N, and C-L ^B-W². In certain embodiments, Z ¹ is CR ⁴. In certain embodiments, Z ¹ is N. In certain embodiments, Z ¹ is C-L ^B-W². In certain embodiments, Z ² is CR ⁴. In certain embodiments, Z ² is N. In certain embodiments, Z ² is C-L ^B-W². In certain embodiments, Z ³ is CR ⁴. In certain embodiments, Z ³ is N. In certain embodiments, Z ³ is C-L ^B-W². In certain embodiments, Z ⁴ is CR ⁴. In certain embodiments, Z ⁴ is N. In certain embodiments, Z ⁴ is C-L ^B-W².

Various combinations of Z ¹、Z²、Z³ and Z ⁴ are possible. For example, in some cases, Z ¹ is CR ⁴,Z² is CR ⁴,Z³ is CR ⁴ and Z ⁴ is CR ⁴. In some cases, Z ¹ is N, Z ² is CR ⁴,Z³ is CR ⁴ and Z ⁴ is CR ⁴. In some cases, Z ¹ is C-L ^B-W²,Z² is CR ⁴,Z³ is CR ⁴ and Z ⁴ is CR ⁴. In some cases, Z ¹ is CR ⁴,Z² is C-L ^B-W²,Z³ is CR ⁴ and Z ⁴ is CR ⁴. In some cases, Z ¹ is CR ⁴,Z² is CR ⁴,Z³ is C-L ^B-W² and Z ⁴ is CR ⁴. In some cases, Z ¹ is CR ⁴,Z² is CR ⁴,Z³ is CR ⁴ and Z ⁴ is C-L ^B-W².

In certain embodiments, R ¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl. In certain embodiments, R ¹ is hydrogen. In certain embodiments, R ¹ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹ is alkynyl or substituted alkynyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ² and R ³ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, 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 linked in a cyclic manner to form a 5 or 6 membered heterocyclyl.

In certain embodiments, R ² is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ² is hydrogen. In certain embodiments, R ² is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ² is methyl. In certain embodiments, R ² is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ² is alkynyl or substituted alkynyl. In certain embodiments, R ² is alkoxy or substituted alkoxy. In certain embodiments, R ² is amino or substituted amino. In certain embodiments, R ² is carboxyl or carboxyl ester. In certain embodiments, R ² is acyl or acyloxy. In certain embodiments, R ² is amido or aminoacyl. In certain embodiments, R ² is an alkylamide or a substituted alkylamide. In certain embodiments, R ² is sulfonyl. In certain embodiments, R ² is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ² is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ² is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ² is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ² is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ³ is hydrogen. In certain embodiments, R ³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³ is methyl. In certain embodiments, R ³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³ is alkynyl or substituted alkynyl. In certain embodiments, R ³ is alkoxy or substituted alkoxy. In certain embodiments, R ³ is amino or substituted amino. In certain embodiments, R ³ is carboxyl or carboxyl ester. In certain embodiments, R ³ is acyl or acyloxy. In certain embodiments, R ³ is amido or aminoacyl. In certain embodiments, R ³ is an alkylamide or a substituted alkylamide. In certain embodiments, R ³ is sulfonyl. In certain embodiments, R ³ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, both R ² and R ³ are methyl.

In certain embodiments, R ² and R ³ are optionally linked in a cyclic manner to form a 5 or 6 membered heterocyclyl. In certain embodiments, R ² and R ³ are cyclic linked to form a 5 or 6 membered heterocyclyl. In certain embodiments, R ² and R ³ are cyclic linked to form a 5 membered heterocyclyl. In certain embodiments, R ² and R ³ are cyclic linked to form a6 membered heterocyclyl.

In certain embodiments, each R ⁴ is independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

The various possibilities for each R ⁴ are described in more detail below. In certain embodiments, R ⁴ is hydrogen. In certain embodiments, each R ⁴ is hydrogen. In certain embodiments, R ⁴ is halogen, such as F, cl, br, or I. In certain embodiments, R ⁴ is F. In certain embodiments, R ⁴ is Cl. In certain embodiments, R ⁴ is Br. In certain embodiments, R ⁴ is I. In certain embodiments, R ⁴ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ⁴ is methyl. In certain embodiments, R ⁴ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ⁴ is alkynyl or substituted alkynyl. In certain embodiments, R ⁴ is alkoxy or substituted alkoxy. In certain embodiments, R ⁴ is amino or substituted amino. In certain embodiments, R ⁴ is carboxyl or carboxyl ester. In certain embodiments, R ⁴ is acyl or acyloxy. In certain embodiments, R ⁴ is amido or aminoacyl. In certain embodiments, R ⁴ is an alkylamide or a substituted alkylamide. In certain embodiments, R ⁴ is sulfonyl. In certain embodiments, R ⁴ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁴ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, R ⁴ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ⁴ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ⁴ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, L ^A is a first linker. Examples of linkers useful in the conjugates of the present disclosure are described in more detail below.

In certain embodiments, L ^B is a second linker. Examples of linkers useful in the conjugates of the present disclosure are described in more detail below.

In certain embodiments, W ¹ is a first drug (or first active agent). Examples of drugs and active agents useful in the conjugates of the present disclosure are described in more detail herein.

In certain embodiments, W ² is a second drug (or second active agent). Examples of drugs and active agents useful in the conjugates of the present disclosure are described in more detail herein.

In certain embodiments, W ³ is a polypeptide (e.g., an antibody). In certain embodiments, W ³ comprises one or more fGly' residues as described herein. In certain embodiments, the polypeptide is attached to the remainder of the conjugate by a fGly' residue described herein. Examples of polypeptides and antibodies useful in the conjugates of the present disclosure are described in more detail herein.

In certain embodiments, the conjugate of formula (I) comprises a first linker L ^A. The first linker L ^A can be used to bind a first moiety of interest (e.g., a first drug or active agent) to a polypeptide (e.g., an antibody) via a conjugate moiety. The first linker L ^A can be bound (e.g., covalently bound) to a conjugate moiety (e.g., as described herein). For example, the first linker L ^A may attach the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety to the first drug or active agent. The hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety may be used to conjugate the first linker L ^A (and thus the first drug or active agent) to a polypeptide, such as an antibody.

For example, as shown in formula (I) above, L ^A is attached to W ³ through a conjugate moiety, and thus W ³ is indirectly bonded to linker L ^A through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. As described above, W ³ is a polypeptide (e.g., an antibody), and thus L ^A is attached to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, e.g., linker L ^A is indirectly bonded to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety.

Any convenient linker may be used for the first linker L ^A in the subject conjugates and compounds. In certain embodiments, the first linker L ^A can comprise a group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, amido, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker L ^A can comprise an alkyl or substituted alkyl group. In certain embodiments, the first linker L ^A can comprise an alkenyl or substituted alkenyl group. In certain embodiments, the first linker L ^A can comprise an alkynyl or substituted alkynyl group. In certain embodiments, the first linker L ^A can comprise an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L ^A can comprise an amino group or a substituted amino group. In certain embodiments, the first linker L ^A can comprise a carboxyl or carboxyl ester group. In certain embodiments, the first linker L ^A can comprise an amido group. In certain embodiments, the first linker L ^A can comprise an alkylamide or a substituted alkylamide group. In certain embodiments, the first linker L ^A can comprise an aryl or substituted aryl group. In certain embodiments, the first linker L ^A can comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L ^A can comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker L ^A can comprise a heterocyclyl or substituted heterocyclyl group.

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

In some embodiments, L ^A is a first linker described by the formula:

-(L¹)_a-(L²)_b-(L³)_c-(L⁴)_d-(L⁵)_e-(L⁶)_f-,

Wherein L ¹、L²、L³、L⁴、L⁵ and L ⁶ are each independently a linker subunit, and a, b, c, d, e and f are each independently 0 or 1.

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

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

Any convenient linker subunit may be used in 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, each of L ¹、L²、L³、L⁴、L⁵ and L ⁶ (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising an alkylene diamine).

In some embodiments, L ¹ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ¹ comprises an aryl group or a substituted aryl group. In some embodiments, L ¹ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ² (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ² comprises an aryl group or a substituted aryl group. In some embodiments, L ² comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ³ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ³ comprises an aryl group or a substituted aryl group. In some embodiments, L ³ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ⁴ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁴ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁴ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ⁵ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁵ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁵ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ⁶ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L ⁶ comprises polyethylene glycol. In some embodiments, L ⁶ comprises a modified polyethylene glycol. In some embodiments, L ⁶ comprises an amino acid residue. In some embodiments, L ⁶ comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁶ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁶ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ^A is a first linker comprising -(L¹)_a-(L²)_b-(L³)_c-(L⁴)_d-(L⁵)_e-(L⁶)_f-, wherein:

- (L ¹)_a -is- (T ¹-V¹)_a -;

- (L ²)_b -is- (T ²-V²)_b -;

- (L ³)_c -is- (T ³-V³)_c -;

- (L ⁴)_d -is- (T ⁴-V⁴)_d -;

- (L ⁵)_e -is- (T ⁵-V⁵)_e -; and

- (L ⁶)_f -is- (T ⁶-V⁶)_f -,

Wherein T ¹、T²、T³、T⁴、T⁵ and T ⁶, if present, are tether groups;

V ¹、V²、V³、V⁴、V⁵ and V ⁶, if present, are covalent bonds or linking functionalities; and

A. b, c, d, e and f are each independently 0 or 1.

As described above, in certain embodiments, L ¹ is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T ¹ is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). In certain embodiments, V ¹ is attached to the first drug or active agent. In certain embodiments, L ², if present, is attached to the first drug or active agent. Thus, in certain embodiments, T ², if present, is attached to the first drug or active agent, or V ², if present, is attached to the first drug or active agent. In certain embodiments, L ³, if present, is attached to the first drug or active agent. Thus, in certain embodiments, T ³, if present, is attached to the first drug or active agent, or V ³, if present, is attached to the first drug or active agent. In certain embodiments, L ⁴, if present, is attached to the first drug or active agent. Thus, in certain embodiments, T ⁴, if present, is attached to the first drug or active agent, or V ⁴, if present, is attached to the first drug or active agent. In certain embodiments, L ⁵, if present, is attached to the first drug or active agent. Thus, in certain embodiments, T ⁵, if present, is attached to the first drug or active agent, or V ⁵, if present, is attached to the first drug or active agent. In certain embodiments, L ⁶, if present, is attached to the first drug or active agent. Thus, in certain embodiments, T ⁶, if present, is attached to the first drug or active agent, or V ⁶, if present, is attached to the first drug or active agent.

In certain embodiments, the conjugate of formula (I) comprises a second linker L ^B. The second linker L ^B can be used to bind a second moiety of interest (e.g., a second drug or active agent) to a polypeptide (e.g., an antibody) via a conjugate moiety. The second linker L ^B can be bound (e.g., covalently bound) to a conjugate moiety (e.g., as described herein). For example, the second linker L ^B may attach the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety to a second drug or active agent. The hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety may be used to conjugate the second linker L ^B (and thus the second drug or active agent) to a polypeptide, such as an antibody.

For example, as shown in formula (I) above, L ^B may be attached to W ³ through a conjugate moiety, and thus W ³ may be indirectly bonded to the second linker L ^B through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. As described above, W ³ is a polypeptide (e.g., an antibody), and thus L ^B may be attached to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, e.g., linker L ^B may be indirectly bonded to the polypeptide (antibody) through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety.

Any convenient linker may be used for the second linker L ^B in the subject conjugates and compounds. In certain embodiments, the second linker L ^B can comprise a group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, amido, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker L ^B can comprise an alkyl or substituted alkyl group. In certain embodiments, the second linker L ^B can comprise an alkenyl or substituted alkenyl group. In certain embodiments, the second linker L ^B can comprise an alkynyl or substituted alkynyl group. In certain embodiments, the second linker L ^B can comprise an alkoxy or substituted alkoxy group. In certain embodiments, the second linker L ^B can comprise an amino group or a substituted amino group. In certain embodiments, the second linker L ^B can comprise a carboxyl or carboxyl ester group. In certain embodiments, the second linker L ^B can comprise an amido group. In certain embodiments, the second linker L ^B can comprise an alkylamide or a substituted alkylamide group. In certain embodiments, the second linker L ^B can comprise an aryl or substituted aryl group. In certain embodiments, the second linker L ^B can comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker L ^B can comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker L ^B can comprise a heterocyclyl or substituted heterocyclyl group.

In certain embodiments, the second linker L ^B may comprise a polymer. For example, the polymer may include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinyl pyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are also possible, as shown by conjugates and compounds described in more detail below.

In some embodiments, L ^B is a second linker described by the formula:

-(L⁷)_g-(L⁸)_h-(L⁹)_i-(L¹⁰)_j-(L¹¹)_k-(L¹²)_l-(L¹³)_m,

Wherein L ⁷、L⁸、L⁹、L¹⁰、L¹¹、L¹² and L ¹³ are each independently a linker subunit, and g, h, i, j, k, L and m are each independently 0 or 1.

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

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

Any convenient linker subunit may be used for 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, each of L ⁷、L⁸、L⁹、L¹⁰、L¹¹、L¹² and L ¹³ (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylene diamines).

In some embodiments, L ⁷ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁷ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁷ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ⁸ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁸ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁸ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ⁹ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L ⁹ comprises polyethylene glycol. In some embodiments, L ⁹ comprises a modified polyethylene glycol. In some embodiments, L ⁹ comprises an amino acid residue. In some embodiments, L ⁹ comprises an alkyl group or a substituted alkyl group. In some embodiments, L ⁹ comprises an aryl group or a substituted aryl group. In some embodiments, L ⁹ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ¹⁰ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ¹⁰ comprises an aryl group or a substituted aryl group. In some embodiments, L ¹⁰ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ¹¹ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ¹¹ comprises an aryl group or a substituted aryl group. In some embodiments, L ¹¹ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ¹² (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L ¹² comprises polyethylene glycol. In some embodiments, L ¹² comprises a modified polyethylene glycol. In some embodiments, L ¹² comprises an amino acid residue. In some embodiments, L ¹² comprises an alkyl group or a substituted alkyl group. In some embodiments, L ¹² comprises an aryl group or a substituted aryl group. In some embodiments, L ¹² comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ¹³ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, 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 comprises an alkyl group or a substituted alkyl group. In some embodiments, L ¹³ comprises an aryl group or a substituted aryl group. In some embodiments, L ¹³ comprises a diamine (e.g., a linking group comprising an alkylene diamine).

In some embodiments, L ^B is a second linker comprising -(L⁷)_g-(L⁸)_h-(L⁹)_i-(L¹⁰)_j-(L¹¹)_k-(L¹²)_l-(L¹³)_m-, wherein:

- (L ⁷)_g -is- (T ⁷-V⁷)_g -;

- (L ⁸)_h -is- (T ⁸-V⁸)_h -;

- (L ⁹)_i -is- (T ⁹-V⁹)_i -;

- (L ¹⁰)_j -is- (T ¹⁰-V¹⁰)_j -;

- (L ¹¹)_k -is- (T ¹¹-V¹¹)_k -;

- (L ¹²)_l -is- (T ¹²-V¹²)_l -; and

- (L ¹³)_m -is- (T ¹³-V¹³)_m -,

Wherein T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³, if present, are tether groups;

V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³, if present, are covalent bonds or linking functionalities; and

G. h, i, j, k, l and m are each independently 0 or 1.

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

As described above, in certain embodiments, L ⁷ is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T ⁷ is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). In certain embodiments, V ⁷ is attached to a second drug or active agent. In certain embodiments, L ⁸, if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ⁸, if present, is attached to a second drug or active agent, or V ⁸, if present, is attached to a second drug or active agent. In certain embodiments, L ⁹, if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ⁹, if present, is attached to a second drug or active agent, or V ⁹, if present, is attached to a second drug or active agent. In certain embodiments, L ¹⁰, if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ¹⁰, if present, is attached to a second drug or active agent, or V10 ⁴, if present, is attached to a second drug or active agent. In certain embodiments, L ¹¹, if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ¹¹, if present, is attached to a second drug or active agent, or V ¹¹, if present, is attached to a second drug or active agent. In certain embodiments, L ¹², if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ¹², if present, is attached to a second drug or active agent, or V ¹², if present, is attached to a second drug or active agent. In certain embodiments, L ¹³, if present, is attached to a second drug or active agent. Thus, in certain embodiments, T ¹³, if present, is attached to a second drug or active agent, or V ¹³, if present, is attached to a second drug or active agent.

With respect to tether groups T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³, any convenient tether group may be used in the subject linker. In some embodiments ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ each comprise one or more groups independently selected from: covalent bond, (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide, and ester, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12.

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises a (C ₁-C₁₂) alkyl group or a substituted (C ₁-C₁₂) alkyl group. In certain embodiments, (C ₁-C₁₂) alkyl is a linear or branched alkyl group comprising 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, (C ₁-C₁₂) alkyl may be alkyl or substituted alkyl, such as C ₁-C₁₂ alkyl, or C ₁-C₁₀ alkyl, or C ₁-C₆ alkyl, or C ₁-C₃ alkyl. In some cases, (C ₁-C₁₂) alkyl is C ₂ -alkyl. For example, (C ₁-C₁₂) alkyl may be alkylene or substituted alkylene, such as C ₁-C₁₂ alkylene, or C ₁-C₁₀ alkylene, or C ₁-C₆ alkylene, or C ₁-C₃ alkylene. In some cases, (C ₁-C₁₂) alkyl is C ₁ -alkylene (e.g., CH ₂). In some cases, (C ₁-C₁₂) alkyl is C ₂ -alkylene (e.g., CH ₂CH₂). In some cases, (C ₁-C₁₂) alkyl is C ₃ -alkylene (e.g., CH ₂CH₂CH₂).

In certain embodiments, substituted (C ₁-C₁₂) alkyl straight or branched substituted alkyl groups include 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, the substituted (C ₁-C₁₂) alkyl may be a substituted alkyl, such as a substituted C ₁-C₁₂ alkyl, or a substituted C ₁-C₁₀ alkyl, or a substituted C ₁-C₆ alkyl, or a substituted C ₁-C₃ alkyl. In some cases, the substituted (C ₁-C₁₂) alkyl is a substituted C ₂ -alkyl. For example, the substituted (C ₁-C₁₂) alkyl group may be a substituted alkylene group, such as a substituted C ₁-C₁₂ alkylene group, or a substituted C ₁-C₁₀ alkylene group, or a substituted C ₁-C₆ alkylene group, or a substituted C ₁-C₃ alkylene group. In some cases, the substituted (C ₁-C₁₂) alkyl is a substituted C ₁ -alkylene (e.g., a C ₁ -alkylene substituted with-SO ₃ H). In some cases, the substituted (C ₁-C₁₂) alkyl is a substituted C ₂ -alkylene. In some cases, the substituted (C ₁-C₁₂) alkyl is a substituted C ₃ -alkylene. For example, a substituted (C ₁-C₁₂) alkyl group can comprise a C ₁-C₁₂ alkylene (e.g., C ₃ -alkylene or C ₅ -alkylene) substituted with a (PEG) _k group as described herein (e.g., -CONH (PEG) _k, such as-CONH (PEG) ₃ or-CONH (PEG) ₅), or-NHCO (PEG) _k, such as-NHCO (PEG) ₇), or can comprise a C ₁-C₁₂ alkylene (e.g., C ₃ -alkylene) substituted with a-CONHCH ₂CH₂SO₃ H group, or can comprise a C ₁-C₁₂ alkylene (e.g., C ₅ -alkylene) substituted with a-NHCOCH ₂SO₃ H group.

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises an aryl or substituted aryl group. For example, the aryl group may be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl may be substituted with one or more substituents selected from the group consisting of (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) 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 substituents include cleavable moieties described herein (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives).

In some cases, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises a heteroaryl or substituted heteroaryl, such as a triazolyl (e.g., 1,2, 3-triazolyl). In some cases, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises a heterocyclyl or substituted heterocyclyl. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl include cleavable moieties described herein (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives).

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises an Ethylenediamine (EDA) moiety, e.g., an EDA-containing tethering group. In certain embodiments, (EDA) _w comprises one or more EDA moieties, such as where w is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1,2, 3,4,5, or 6. The attached Ethylenediamine (EDA) moiety may optionally be substituted at one or more convenient positions with any convenient substituent such as alkyl, substituted alkyl, acyl, substituted acyl, aryl or substituted aryl. In certain embodiments, the EDA portion is described by the following structure:

Wherein y is an integer from 1 to 6, or is 0 or 1, and each R ¹² is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, 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 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R ¹² groups of EDA may be joined ring-wise, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R ¹² groups are alkyl groups, the rings of which are joined to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R ¹² groups are selected from hydrogen, alkyl (e.g., methyl), and substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises a 4-amino-piperidine (4 AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be optionally substituted at one or more convenient positions with any convenient substituent, for example, with an alkyl group, a substituted alkyl group, a polyethylene glycol moiety, an acyl group, a substituted acyl group, an aryl group, or a substituted aryl group. In some embodiments, the 4AP portion is described by the following structure:

Wherein R ¹² is selected from the group consisting of 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 ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹² is a polyethylene glycol moiety. In certain embodiments, R ¹² is a carboxyl-modified polyethylene glycol.

In certain embodiments, R ¹² comprises a polyethylene glycol moiety described by formula (PEG) _k, which can be represented by the following structure:

Where k is an integer from 1 to 20, such as1 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, such as1, 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, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹⁷ is COOH. In certain embodiments, R ¹⁷ is OH. In certain embodiments, R ¹⁷ is OCH ₃.

In certain embodiments, the tether group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises (PEG) _n, wherein (PEG) _n is polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG) _n is described by the following structure:

Wherein n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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.

In certain embodiments, the tether group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises (AA) _p, wherein AA is an amino acid residue. Any convenient amino acid may be used. Amino acids of interest include, but are not limited to, L-and D-amino acids, naturally occurring amino acids (such as any of 20 primary alpha-amino acids and beta-alanine), non-naturally occurring amino acids (e.g., amino acid analogs) (such as non-naturally occurring alpha-amino acids or non-naturally occurring beta-amino acids, etc.). In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

In certain embodiments, the tether groups (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) include amino acid analogs. Amino acid analogs include compounds that are similar in structure and/or overall shape to one or more amino acids typically found in naturally occurring proteins (e.g., ala or A, cys or C, asp or D, glu or E, phe or F, gly or G, his or H, ile or I, lys or K, leu or L, met or M, asn or N, pro or P, gln or Q, arg or R, ser or S, thr or T, val or V, trp or W, tyr or Y). Amino acid analogs also include natural amino acids having modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D-form, as well as amino acid analogs in the L-form. In some cases, amino acid analogs share the backbone structure and/or side chain structure of one or more natural amino acids, except for one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (e.g., N) for the relevant atom (e.g., S), addition of a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., cl or Br, etc.), deletion of a group, substitution of a covalent bond (e.g., substitution of a single bond for a double bond, etc.), 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, cysteic acid, and the like.

In certain embodiments, tethering groups (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) include moieties described by the formula- (CR ¹³OH)_x -wherein x is 0 or x is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, R ¹³ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxy ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, in certain embodiments, R ¹³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl, in certain embodiments, R ¹³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl, in certain embodiments, R ¹³ is alkynyl or substituted alkynyl, r ¹³ is alkoxy or substituted alkoxy. In certain embodiments, R ¹³ is amino or substituted amino. In certain embodiments, R ¹³ is carboxyl or carboxyl ester. In certain embodiments, R ¹³ is acyl or acyloxy. In certain embodiments, R ¹³ is amido or aminoacyl. In certain embodiments, R ¹³ is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹³ is sulfonyl. In certain embodiments, R ¹³ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹³ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ¹³ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R ¹³.

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises an acetal group, disulfide, hydrazine, or ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

In certain embodiments, the tethering group (e.g., ,T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³) comprises m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), or p-hydroxy-phenyl (PHP).

In some embodiments, the tethering group comprises a MABO group described by the following structure:

in some embodiments, the tethering group comprises a MABC group described by the following structure:

/>

in some embodiments, the tethering group comprises a PABO group described by the following structure:

in some embodiments, the tethering group comprises a PABC group described by the following structure:

in some embodiments, the tethering group comprises a PAB group described by the following structure:

In some embodiments, the tether group comprises a PABA group described by the following structure:

in some embodiments, the tether group comprises a PAP group described by the following structure:

in some embodiments, the tethering group comprises a PHP group described by the following structure:

In certain embodiments, each R ¹⁴ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R ¹⁴ is hydrogen. In certain embodiments, each R ¹⁴ is hydrogen. In certain embodiments, R ¹⁴ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹⁴ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹⁴ is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁴ is alkoxy or substituted alkoxy. In certain embodiments, R ¹⁴ is amino or substituted amino. In certain embodiments, R ¹⁴ is carboxyl or carboxyl ester. In certain embodiments, R ¹⁴ is acyl or acyloxy. In certain embodiments, R ¹⁴ is amido or aminoacyl. In certain embodiments, R ¹⁴ is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹⁴ is sulfonyl. In certain embodiments, R ¹⁴ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁴ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹⁴ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹⁴ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹⁴ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In some embodiments of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tethering structures shown above, the benzene ring may be substituted with one or more additional groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, one or more of the tethering groups T¹、T²、T³、T⁴、T⁵、T⁶、T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and/or T ¹³ are each optionally substituted with a glycoside or glycoside derivative. For example, in some cases, T ¹、T²、T³、T⁴、T⁵ and T ⁶ are each optionally substituted with a glycoside. In some cases, T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each optionally substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

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

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

With respect to linking functionalities V¹、V²、V³、V⁴、V⁵、V⁶、V⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³, any convenient linking functionality may be used for the subject linker. Linking functionalities of interest include, but are not limited to, amino, carbonyl, amino, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonamido, aminosulfonyl, thio, oxy, phosphate (phospho), phosphoramido, thiophosphoramido, and the like. In some embodiments ,V¹、V²、V³、V⁴、V⁵、V⁶、V⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ are each independently selected from the group consisting of covalent bonds 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1,2, 3, 4,5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

In some embodiments, each R ¹⁵ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, amido, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R ¹⁵ is hydrogen. In certain embodiments, each R ¹⁵ is hydrogen. In certain embodiments, R ¹⁵ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹⁵ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹⁵ is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁵ is alkoxy or substituted alkoxy. In certain embodiments, R ¹⁵ is amino or substituted amino. In certain embodiments, R ¹⁵ is carboxyl or carboxyl ester. In certain embodiments, R ¹⁵ is acyl or acyloxy. In certain embodiments, R ¹⁵ is amido or aminoacyl. In certain embodiments, R ¹⁵ is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹⁵ is sulfonyl. In certain embodiments, R ¹⁵ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁵ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹⁵ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹⁵ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹⁵ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, each R ¹⁵ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 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 ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R ¹⁵.

As described above, in some embodiments, L ^A is a first linker comprising -(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e-(T⁶-V⁶)_f-, wherein a, b, c, d, e and f are each independently 0 or 1.

In some embodiments, in the first linker L ^A:

T ¹ is selected from (C ₁-C₁₂) alkyl and substituted (C ₁-C₁₂) alkyl;

T ²、T³、T⁴、T⁵ and T ⁶ are each independently selected from (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, disulfide, hydrazine and esters; and

V ¹、V²、V³、V⁴、V⁵ and V ⁶ are each independently selected from the group consisting of covalent bonds 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein q is an integer from 1 to 6;

wherein:

(PEG) _n is Wherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:

Wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) is

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

Each R ¹² is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R ¹² groups may be joined ring-wise to form a piperazinyl ring;

Each R ¹³ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, T ¹、T²、T³、T⁴、T⁵ and T ⁶ and V ¹、V²、V³、V⁴,V⁵ and V ⁶ are selected from the following:

wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CO-;

T ² is an amino acid analog and V ² is-NH-;

T ³ is (PEG) _n and V ³ is-CO-;

T ⁴ is AA and V ⁴ is absent;

T ⁵ is PABC and V ⁵ is absent; and

F is 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is (PEG) _n and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is substituted (C ₁-C₁₂) alkyl and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0.

In certain embodiments, the left-hand side of the above linker structure for the first linker L ^A is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, and the right-hand side of the above linker structure for the first linker L ^A is attached to the first drug or active agent.

As described above, in some embodiments, L ^B is a second linker comprising -(T⁷-V⁷)_g-(T⁸-V⁸)_h-(T⁹-V⁹)_i-(T¹⁰-V¹⁰)_j-(T¹¹-V¹¹)_k-(T¹²-V¹²)_l-(T¹³-V¹³)_m-, wherein g, h, i, j, k, L and m are each independently 0 or 1.

In some embodiments, in the second linker L ^B:

t ⁷ is selected from (C ₁-C₁₂) alkyl and substituted (C ₁-C₁₂) alkyl;

T ⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each independently selected from (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, disulfide, hydrazine, and esters; and

V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ are each independently selected from the group consisting of covalent bonds 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein q is an integer from 1 to 6;

wherein:

(PEG) _n is Wherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:

Wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) is

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

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

Any convenient tethering group may be used for T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³. For example, any of the tethering groups described above with respect to T ¹、T²、T³、T⁴、T⁵ and T ⁶ may be used for tethering groups T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³.

Any convenient linking functionality may be used for V ⁷、V⁸、V⁹、V¹⁰,V¹¹、V¹² and V ¹³. For example, any of the linking functionalities described above with respect to V ¹、V²、V³、V⁴、V⁵ and V ⁶ may be used to link the functionalities V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³.

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

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

In certain embodiments of the second linker L ^B, one or more of the tether groups T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In certain embodiments of the second linker L ^B, the MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above, the benzene ring may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In certain embodiments, T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ and V ⁷、V⁸、V⁹、V¹⁰,V¹¹、V¹² and V ¹³ are selected from the following:

wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is (PEG) _n and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent; and

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is substituted (C ₁-C₁₂) alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent;

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0.

In certain embodiments, the left-hand side of the above linker structure for the second linker L ^B is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, and the right-hand side of the above linker structure for the second linker L ^B is attached to a second drug or active agent.

In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and one or more drugs or active agents are linked together 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 comprising one or more cleavable moieties, wherein the cleavable moiety comprises one or more bonds that are cleavable under certain conditions, thereby separating the cleavable linker into two or more separable moieties. For example, a cleavable moiety may comprise one or more covalent bonds that, under certain conditions, can be dissociated or broken to separate the cleavable linker into two or more moieties. Thus, the linker included in the antibody-drug conjugate may be a cleavable linker such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at the desired action target site of the drug.

In some cases, the cleavable linker comprises two cleavable moieties, e.g., a first cleavable moiety and a second cleavable moiety. The cleavable moiety may be configured such that cleavage of both cleavable moieties is required in order to separate or release the drug from the antibody at the desired site of action target of the drug. For example, cleavage of a cleavable linker may 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 blocks cleavage of the first cleavable moiety. By "hindering cleavage" it is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage of the first cleavable moiety, thereby substantially reducing the amount of cleavable linker or preventing cleavage of the cleavable linker. For example, the presence of an uncleaved second cleavable moiety may hinder cleavage of the first cleavable moiety. The presence of the second cleavable moiety prevents cleavage of the first cleavable moiety, in turn significantly reduces the amount of drug released from the antibody or prevents release of drug from the antibody. For example, premature release of the drug from the antibody can be significantly reduced or prevented until the antibody-drug conjugate is at or near the desired action target site of the drug.

In some cases, cleavage of the cleavable linker may be achieved by initially cleaving the second cleavable moiety and then cleaving the first cleavable moiety, as the second cleavable moiety blocks cleavage of the first cleavable moiety. Cleavage of the second cleavable moiety may reduce or eliminate the obstruction of cleavage of the first cleavable moiety, thereby allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety may result in cleavage or separation of the cleavable linker into two or more moieties as described above to release the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of the uncleaved second cleavable moiety. By substantially is meant that about 10% or less of the cleavage of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety, 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 of the cleavage of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety.

In other words, the second cleavable moiety may 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 and thus significantly reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired action target site of the drug. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects (deprotect) the first cleavable moiety), allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which in turn separates or releases the drug from the antibody at the desired site of action target of the drug as described above. In some cases, cleavage of the second cleavable moiety exposes the first cleavable moiety for subsequent cleavage, but 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 still required to cleave the cleavable linker).

The cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety. For example, the first cleavable moiety may be a first enzymatically cleavable moiety and the second cleavable moiety may be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above by enzymatic action of an enzyme. The enzymatically cleavable moiety may be any cleavable moiety cleavable by enzymatic action of an enzyme such as, but not limited to, esters, peptides, glycosides, and the like. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at a desired action target site, such as a desired action target site of a drug to be released from an antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in a significant amount in other areas (e.g., whole blood, plasma, or serum). Thus, cleavage of the enzymatically cleavable moiety may be controlled such that substantial cleavage occurs at the desired site of action, while cleavage does not occur significantly in other regions or until the antibody-drug conjugate reaches the desired site of action.

For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, such as for delivering a cancer therapeutic drug to a desired site of action in the presence of cancer cells. In some cases, an enzyme, such as an esterase that cleaves an ester bond or a glycosidase that cleaves a glycosidic bond, can be a biomarker for cancer that is overexpressed in cancer cells. Overexpression, and thus localization, of certain enzymes in cancer can be used in the context of enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (i.e., the site of cancer (and overexpressed enzymes)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or glycoside) that is cleavable by an enzyme that is overexpressed in a cancer cell. For example, the enzyme may be an esterase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that is cleavable by an esterase. In some cases, the enzyme may be a glycosidase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that is cleavable by a glycosidase.

In certain embodiments, the enzymatically cleavable moiety is an ester bond. For example, the first cleavable moiety described above (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can comprise an ester. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by esterases and thereby significantly reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired site of action target of the drug. In some cases, a portion of the linker adjacent to the first cleavable moiety is linked to or includes a substituent, wherein the substituent includes the second cleavable moiety. In some cases, the second cleavable moiety comprises a glycoside or glycoside derivative.

In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycosyl) or glycoside derivative. In some cases, the glycoside or glycoside derivative may promote an increase in hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside or glycoside derivative. The glycoside or glycoside derivative may be any glycoside or glycoside derivative suitable for use in a cleavable linker and cleavable by enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable moiety comprises an ester and the second cleavable moiety comprises a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from the group consisting of 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.

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

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

Any of the chemical entities, linkers, and conjugate moieties described in the structures above may be suitable for use with the subject compounds and conjugates.

Additional disclosures relating to hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds, and methods of making conjugates, are found in U.S. patent No. 9,310,374 and U.S. patent No. 9,493,413, the disclosures of each of which are incorporated herein by reference. Additional disclosures relating to cleavable linkers are found in WO 2020/154437 filed on month 1 and 22 of 2020, PCT/US2021/060193 filed on month 11 and 19 of 2022, and PCT/US2022/012347 filed on month 1 and 13 of 2022, the disclosures of which are incorporated herein by reference. Additional disclosures relating to branching linkers are found in WO 2020/154437 filed on month 1 and 22 of 2020, PCT/US2021/060193 filed on month 11 and 19 of 2022 and PCT/US2022/018534 filed on month 3 and 2 of 2022, the disclosures of which are incorporated herein by reference.

Compounds useful for producing conjugates

The present disclosure provides compounds useful for producing conjugates described herein. In certain embodiments, the compounds may be attached to one or more drugs or active agents, and may further include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties useful for conjugating one or more drugs or active agents to a polypeptide (e.g., an antibody). For example, a conjugate moiety in a compound may be conjugated to a polypeptide (e.g., an antibody) such that one or more drugs or active agents are indirectly bound to the polypeptide (antibody).

In certain embodiments, the compound is a compound of formula (II):

wherein:

l ^A is a first linker;

L ^B is a second linker;

W ¹ is a first drug; and

W ² is a second drug.

With respect to the compound of formula (II), substituents Z¹、Z²、Z³、Z⁴、R²、R³、R⁴、L^A、L^B、W¹ and W ² are as described above with respect to the conjugate of formula (I). Similarly, the substituents for the first and second linkers L ^A and L^B,T¹、T²、T³、T⁴、T⁵、T⁶、V¹、V²、V³、V⁴、V⁵ and V ⁶ and T⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹²、T¹³、V⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ of formula (II) are as described above for the conjugates of formula (I).

For example, in some cases, T ¹、T²、T³、T⁴、T⁵ and T ⁶, and V ¹、V²、V³、V⁴、V⁵ and V ⁶ are selected from the following:

wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CO-;

T ² is an amino acid analog and V ² is-NH-;

T ³ is (PEG) _n and V ³ is-CO-;

T ⁴ is AA and V ⁴ is absent;

T ⁵ is PABC and V ⁵ is absent; and

F is 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is (PEG) _n and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is substituted (C ₁-C₁₂) alkyl and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0.

For example, in some cases, T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³, and V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ are selected from the following:

wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is (PEG) _n and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent; and

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is substituted (C ₁-C₁₂) alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent;

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0.

The compounds of formula (II) may be used in the conjugation reactions described herein, wherein one or more drugs or active agents attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety are conjugated with a polypeptide (e.g., an antibody) to form an antibody-drug conjugate.

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

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Anti-Nectin-4 antibodies

As described above, the subject conjugates may comprise an anti-Nectin-4 antibody as substituent W ², wherein the amino acid sequence of the anti-Nectin-4 antibody has been modified to comprise a 2-formylglycine (fGly) residue. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and/or their standard one-letter abbreviations, such as: alanine or Ala or a; cysteine or Cys or C; aspartic acid 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; aspartic acid or Asn or N; proline or Pro or P; glutamine or gin 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.

The various protein sequences identified in the present disclosure are provided in table 2 below and are referenced in their entirety based on their sequence identifiers (SEQ ID No.).

Table 2. Variable Heavy (VH) and Variable Light (VL) sequences and sequence identifiers of the various antibodies disclosed herein. The first, second and third CDR sequences in each VH and VL sequence are shown in bold and underlined. The first framework (FW 1) region is before the first CDR, the second framework (FW 2) region is after the first CDR, the third framework (FW 3) region is after the second CDR, and the fourth framework (FW 4) region is after the third CDR.

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CDR sequences and corresponding sequence identifiers are provided in table 3 below:

table 3: CDR sequences of the various VH and VL chains disclosed herein.

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According to some embodiments, the antibodies of the disclosure specifically bind to Nectin-4 and comprise:

a variable heavy chain (VH) chain comprising heavy chain CDRs 1-3 (HCDR 1-3) of a VH chain having a sequence selected from SEQ ID NOs 1 to 17; and

A variable light chain (VL) chain comprising light chain CDR 1-3 (LCDR 1-3) of a VL chain having a sequence selected from SEQ ID NOs 18 to 31.

a VH chain comprising a sequence selected from SEQ ID NOs 1 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 31.

In some cases, the antibodies of the disclosure specifically bind to Nectin-4 and comprise:

a VH chain comprising HCDR 1-3 having a VH chain with a sequence selected from SEQ ID NOs 1 to 6; and

A VL chain comprising LCDR 1-3 of a VL chain having a sequence selected from SEQ ID NOs 18 to 23.

Thus, in certain embodiments, the antibodies of the disclosure specifically bind to Nectin-4 and comprise:

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

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 23.

Antibodies comprising any combination of heavy and light chains as shown in table 4 below are contemplated.

Table 4. Combinations of HV and HL chains in certain embodiments of the invention.

A VH chain comprising HCDR 1-3 having a VH chain with a sequence selected from SEQ ID NOs 7 to 13; and

A VL chain comprising LCDR 1-3 of a VL chain having a sequence selected from SEQ ID NOs 24 to 27.

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

A VL chain comprising a sequence selected from SEQ ID NOs 24 to 27.

In certain embodiments, antibodies comprising any combination of heavy and light chains as set forth in table 5 below are contemplated.

Table 5. Combinations of HV and HL chains in certain embodiments of the invention.

a VH chain comprising HCDR 1-3 of a VH chain having the sequence of SEQ ID NO. 14; and

A VL chain comprising LCDR 1-3 of a VL chain having the sequence of SEQ ID No. 28.

A VH chain comprising the sequence of SEQ ID NO. 14; and

A VL chain comprising the sequence of SEQ ID NO. 28.

A VH chain comprising HCDR 1-3 of a VH chain having the sequence of SEQ ID NO. 15; and

A VL chain comprising LCDR 1-3 of a VL chain having the sequence of SEQ ID NO. 29.

a VH chain comprising the sequence of SEQ ID NO. 15; and

A VL chain comprising the sequence of SEQ ID NO. 29.

a VH chain comprising HCDR 1-3 of a VH chain having the sequence of SEQ ID No. 16; and

A VL chain comprising LCDR 1-3 of a VL chain having the sequence of SEQ ID No. 30.

a VH chain comprising the sequence of SEQ ID No. 16; and

A VL chain comprising the sequence of SEQ ID NO. 30.

A VH chain comprising HCDR 1-3 of a VH chain having the sequence of SEQ ID No. 17; and

A VL chain comprising LCDR 1-3 of a VL chain having the sequence of SEQ ID NO. 31.

a VH chain comprising the sequence of SEQ ID No. 17; and

A VL chain comprising the sequence of SEQ ID NO. 31.

In certain embodiments, the VH chain of the anti-Nectin-4 antibody comprises an HCDR 1-3 having a VH chain selected from the group consisting of sequences SEQ ID NOS.1-17 and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence set forth in the sequence selected from the group consisting of sequences SEQ ID NOS.1-17. In certain embodiments, any amino acid differences between the VH chain of the anti-Nectin-4 antibodies of the disclosure and the sequence selected from SEQ ID nos. 1 to 17 may be limited to regions outside the CDRs, for example within a Framework Region (FR), for example one or more of FR1, FR2, FR3 and/or FR 4.

In certain embodiments, the VL chain of an anti-Nectin-4 antibody comprises LCDR 1-3 of a VL chain having a sequence selected from the group consisting of SEQ ID NOS: 18 to 31 and comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to the amino acid sequence set forth in the sequence selected from the group consisting of SEQ ID NOS: 18 to 31.

In certain embodiments, any amino acid differences between the VL chain of an anti-Nectin-4 antibody of the disclosure and sequences selected from SEQ ID NOS: 18 to 31 may be limited to regions outside the CDRs, for example within one or more of FR1, FR2, FR3 and/or FR 4.

In certain embodiments, an anti-Nectin-4 antibody of the disclosure may comprise: a) A heavy chain comprising a VH region having an amino acid sequence shown in a sequence selected from SEQ ID NOs 1 to 17 and a heavy chain constant region having an amino acid sequence shown in any one of SEQ ID NOs 70 to 86 wherein C present in sequence LCTPSR in the constant region is replaced with fGly; and b) a light chain comprising a VL region having an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NOS.18 to 31.

In certain embodiments, an anti-Nectin-4 antibody of the disclosure may comprise: a) A heavy chain comprising a VH region comprising an amino acid sequence that is at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical) to an amino acid sequence set forth in a sequence selected from SEQ ID nos. 1 to 17, and a heavy chain constant region comprising an amino acid sequence that is at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to an amino acid sequence set forth in any one of SEQ ID nos. 70 to 86, wherein the C present in sequence LCTPSR in the constant region is replaced with fGly ', wherein fGly' refers to an amino acid residue conjugated to a moiety of interest; and b) a light chain comprising a VL region comprising an amino acid sequence that is at least 85% identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical) to an amino acid sequence set forth in a sequence selected from the group consisting of SEQ ID nos. 18 to 31.

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

The anti-Nectin-4 antibodies of the present disclosure can bind to cancerous tissue and can exhibit no binding or insignificant binding to normal tissue (e.g., insignificant binding as measured by immunohistochemistry or binding undetectable by immunohistochemistry). For example, the anti-Nectin-4 antibodies described herein can bind to human solid tumors having cancer cells, such as ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer, while exhibiting no detectable binding to human normal tissues (such as ovaries, breasts, lungs, and pancreas without cancer cells).

Antibodies find use in a variety of research, diagnostic, and therapeutic applications, including for performing any of the methods described in U.S. patent application nos. 20210130459, 20200231670, 20180243434, 20110301056, 20100285597, 20080268476 (the disclosures of each of which are incorporated herein by reference in their entirety).

The subject antibodies specifically bind to a Nectin-4 polypeptide, wherein the epitope comprises amino acid residues within a human Nectin-4 antigen comprising the amino acid sequence set forth in SEQ ID NO. 99:

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.

The subject antibodies exhibited high affinity binding to Nectin-4. For example, the subject antibody binds 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 greater than 10 ^-12 M. The subject antibodies bind to the epitope present on Nectin-4 with an affinity of about 10 ^-7 M to about 10 ^-8 M, about 10 ^-8 M to about 10 ^-9 M, about 10 ^-9 M to about 10 ^-10 M, about 10 ^- ¹⁰ M to about 10 ^-11 M, or about 10 ^-11 M to about 10 ^-12 M, or greater than 10 ^-12 M.

The anti-Nectin-4 antibodies of the disclosure may, in some cases, induce apoptosis in cells whose cell surface expresses Nectin-4.

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.

The term "immunoglobulin" as used herein refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA 1 and IgA 2), gamma (IgG 1, igG2, igG3, igG 4), delta, epsilon, and mu constant region genes; and many immunoglobulin variable region genes. The full length immunoglobulin light chain (about 25kD or 214 amino acids) is encoded by a variable region gene (about 110 amino acids) at the N-terminus and a kappa or lambda constant region at the C-terminus. The full length immunoglobulin heavy chain (about 50kD or 446 amino acids) is encoded by a variable region gene (about 116 amino acids) at the N-terminus and one of the other above-described constant region genes at the C-terminus, e.g., gamma (encoding about 330 amino acids). In some embodiments, the subject antibodies include a full length immunoglobulin heavy chain and a full length immunoglobulin light chain.

In some embodiments, the subject antibodies do not include a full length immunoglobulin heavy chain and a full length immunoglobulin light chain, but rather include antigen binding fragments of a full length immunoglobulin heavy chain and a full length immunoglobulin light chain. In some embodiments, the antigen binding fragments are contained on separate polypeptide chains; in other embodiments, the antigen binding fragments are contained within a single polypeptide chain. As described above, the term "antigen binding fragment" refers to one or more fragments of a full length antibody that are capable of specifically binding to Nectin-4. Examples of binding fragments include (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains); (ii) A F (ab') ₂ fragment (bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region); (iii) Fd fragment (consisting of VH and CH1 domains); (iv) Fv fragments (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of a VH domain); (vi) isolated CDRs; (vii) Single chain Fv (scFv) (consisting of VH and VL domains of a single arm of an antibody linked by a synthetic linker using recombinant methods such that the VH and VL domains pair to form a monovalent molecule); (viii) Diabodies (consisting of two scfvs, wherein the VH and VL domains are linked such that they do not pair to form monovalent molecules; the VH of each scFv paired with the VL domain of the other scFv to form a bivalent molecule); (ix) Bispecific antibodies (consisting of at least two antigen binding regions, each region binding a different epitope). In some embodiments, the subject antibody fragment is a Fab fragment. In some embodiments, the subject antibody fragment is a single chain antibody (scFv).

In some embodiments, the subject antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized, or in vitro generated antibody. The term "recombinant" or "modified" antibody as used herein is intended to include all antibodies produced, expressed, produced, or isolated by recombinant methods, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant, combinatorial antibody library; (iii) Antibodies isolated from human immunoglobulin gene transgenic animals (e.g., mice); or (iv) antibodies produced, expressed, produced or isolated by any other method involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized and in vitro generated antibodies; and may optionally include constant regions derived from human germline immunoglobulin sequences.

Full length bispecific antibodies can be generated as follows: for example, fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies is used, by introducing substitutions at the heavy chain CH3 interface in each half-molecule, to facilitate heterodimer formation of two antibody half-molecules with different specificities in an in vitro cell-free environment or using co-expression of one of the two. Fab arm exchange reactions are the result of disulfide isomerization reactions and dissociation-association of CH3 domains. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The free cysteine of one of the resulting parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine residue of the second parent monospecific antibody molecule, and at the same time the CH3 domain of the parent antibody is released and recombined by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a different epitope.

A "knob-in-hole" strategy (see, e.g., PCT International publication No. WO 2006/028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the CH3 domain interface in human IgG may be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (mortar) are introduced into the heavy chain of an antibody that specifically binds a first antigen, and amino acids with large side chains (pestle) are introduced into the heavy chain of an antibody that specifically binds a second antigen. After co-expression of the two antibodies, heterodimers are formed due to preferential interactions of the heavy chain with a "mortar" with the heavy chain with a "pestle". An exemplary CH3 substitution pair forming a pestle and mortar is (expressed as modification position in the first CH3 domain of the first heavy chain/modification position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F W/Y407A, T394W/Y407T, T3945/Y407A, T366W/T394S, F W/T394S and T366W/T366S/L368A/Y407V.

Other strategies may be used, such as using electrostatic interactions to promote heavy chain heterodimerization by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface, as in US patent publication No. US2010/0015133; US patent publication No. US2009/0182127; described in US patent publication No. US2010/028637 or US patent publication No. US 2011/0123032. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modification positions in the first CH3 domain of the first heavy chain/modification positions in the second CH3 domain of the second heavy chain): L351 Y/F405A/Y407V/T394W、T366I/K392M/T394W/F405A/Y407V、T366L/K392M/T394W/F405A/Y407V、L351Y/Y407A/T366A/K409F、L351Y/Y407A/T366V/K409F、Y407A/T366A/K409F、 or T350V/L351Y/F405A/Y407V, T V/T366L/K392L/T394W as described in US patent publication No. US2012/0149876 or US patent publication No. US 2013/0195849.

Single chain bispecific antibodies are also provided. In some embodiments, the single chain bispecific antibody of the present disclosure is a bispecific scFv. The subject antibodies may be humanized. The constant region, if present, may also be derived substantially or entirely from human immunoglobulins.

Methods for preparing humanized antibodies are known in the art. Substitution of the mouse CDRs into a human variable domain framework can result in preserving their correct spatial orientation, e.g., where the human variable domain framework adopts the same or similar conformation as the mouse variable framework from which the CDRs were derived. This can be achieved by obtaining the human variable domain from a human antibody whose framework sequence exhibits a high degree of sequence identity to the murine variable framework domain from which the CDR is derived. The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. The human antibody sequence may be the sequence of a naturally occurring human antibody or may be a consensus sequence of several human antibodies.

After determining the complementarity determining regions of the murine donor immunoglobulin and the appropriate human acceptor immunoglobulin, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. In general, substitution of murine amino acid residues with human should be minimized because the introduction of murine residues in humans increases the risk of the antibody eliciting a human anti-mouse antibody (HAMA) response. A field-approved method of determining immune responses may be performed to monitor HAMA responses in a particular patient or during a clinical trial. Patients administered humanized antibodies can be given immunogenicity assessment at the beginning of the therapy and throughout the administration. For example, HAMA response is measured by detecting antibodies to a humanized therapeutic agent in a serum sample from a patient using methods known to those skilled in the art, including surface plasmon resonance (BIACORE) and/or solid phase ELISA assays. In many embodiments, the subject humanized antibody does not substantially elicit a HAMA response in a human subject.

Certain amino acids from human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and/or binding to antigen. Unnatural juxtaposition of murine CDR regions with human variable framework regions can lead to unnatural conformational constraints that result in loss of binding affinity unless corrected by substitution of certain amino acid residues. The selection of amino acid residues for substitution can be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Typically, the molecular model is generated starting from the resolved structure of the immunoglobulin chain or domain thereof. The amino acid sequence similarity of the strand to be modeled and the strand or domain of the resolved three-dimensional structure is compared and the strand or domain that shows the greatest sequence similarity is selected as the starting point for the molecular model construction. Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably those sharing at least 60%, 70%, 80%, 90% sequence identity or higher are selected for modeling. The resolved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain being modeled and those in the starting structure. The modified structure is then assembled into a composite immunoglobulin. Finally, the model is improved by energy minimization and by verifying that all atoms are within the proper distance from each other and that bond length and bond angle are within chemically acceptable limits.

When the framework residues (as defined by Kabat, supra) constitute structural loop residues (as defined by Chothia, supra), the amino acids present in the mouse antibody may be selected for substitution of the humanized antibody. Residues "adjacent to CDR regions" include amino acid residues in the primary sequence of the humanized immunoglobulin chain that are immediately adjacent to one or more CDRs, e.g., in the vicinity of a CDR as defined by Kabat, or in the vicinity of a CDR as defined by Chothia (see, e.g., chothia and Lesk JMB 196:901 (1987)). These amino acids may specifically interact with amino acids in the CDRs and, if selected from the acceptor, distort the donor CDR and reduce affinity. In addition, adjacent amino acids may interact directly with the antigen (Amit et al, science,233:747 (1986)), and it may be desirable to select these amino acids from the donor to maintain contact with all antigens that provide affinity in the original antibody.

In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibody is a scFv dimer (e.g., comprising two tandem scFv (scFv ₂)), a scFv trimer (e.g., comprising three tandem scFv (scFv ₃)), a scFv tetramer (e.g., comprising four tandem scFv (scFv ₄)), or a multimer of more than four scFv (e.g., in tandem). scFv monomers can be connected in series via a linker of about 2 amino acids to about 10 amino acids in length, e.g., 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, or 10aa in length. Suitable linkers include, for example, (Gly) _x, where x is an integer from 2 to 10, glycine-serine polymers and the like.

In some embodiments, the subject antibodies include a constant region (e.g., an Fc region) of an immunoglobulin. The Fc region, if present, may be a human Fc region. If constant regions are present, antibodies may comprise light and heavy chain constant regions. Antibodies described herein include antibodies having constant regions of all types, including IgM, igG, igD, igA and IgE, as well as any isotype, including IgG1, igG2, igG3, and IgG4. An example of a suitable heavy chain Fc region is human isotype IgG1 Fc. The light chain constant region may be lambda or kappa. A subject antibody (e.g., a subject humanized antibody) can include sequences from more than one class or isotype. Antibodies may be represented as tetramers comprising two light chains and two heavy chains, as separate heavy chains, as Fab, fab ', F (ab') 2, and Fv, or as single chain antibodies, wherein the heavy and light chain variable domains are linked by a spacer region.

In some embodiments, an anti-Nectin-4 antibody of the disclosure may include one or more amino acid substitutions in the Fc region that introduce the Fc region. In some embodiments, one or more amino acid substitutions may be at positions 239, 298, 326, 330, and 332 in the Fc region. In some embodiments, an anti-Nectin-4 antibody of the disclosure may include one or more of the following amino acid substitutions introduced into the Fc region: I332E; S239D/A330L/I332E; S239D/S298A/I332E; S239D/K326T/I332E; S239D/S298A/K326T/I332E; or S239D/A330L/I332E/D356E/L358M.

In some embodiments, the subject antibodies include a free thiol (-SH) group at the carboxy terminus, wherein the free thiol can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), scaffold, carrier, and the like.

In some embodiments, the subject antibodies comprise one or more non-naturally occurring amino acids. In some embodiments, the non-naturally encoded amino acid comprises carbonyl, acetyl, aminooxy, hydrazino, hydrazono, semicarbazide, azide, or alkyne groups. The inclusion of non-naturally occurring amino acids can provide for attachment to a polymer, a second polypeptide, a scaffold, or the like. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine.

The present disclosure also provides anti-Nectin-4 antibodies having an attachment moiety of interest (e.g., a detectable label, a drug, a half-life extending moiety, etc.). Modification of antibodies can be accomplished by various synthetic and/or recombinant methods. The moiety or moieties attached to the antibody may provide one or more of a variety of functions or features. Exemplary moieties include detectable labels (e.g., dye labels (e.g., chromophores, fluorophores), biophysical probes (spin labels, nuclear Magnetic Resonance (NMR) probes), fluorescence Resonance Energy Transfer (FRET) type labels (e.g., at least one member of a FRET pair, including at least one member of a fluorophore/quencher pair), bioluminescence Resonance Energy Transfer (BRET) type labels (e.g., at least one member of a BRET pair), immunodetectable labels (e.g., FLAG, his (6), etc.), water-soluble polymers (e.g., pegylation), purification labels (e.g., to facilitate separation by affinity chromatography (e.g., attachment of a FLAG epitope), membrane localization domains (e.g., lipid or Glycosyl Phosphatidylinositol (GPI) type anchoring), immobilization labels (e.g., to facilitate attachment of polypeptides to surfaces, including selective attachment, including drug targeting (e.g., to facilitate attachment of drugs to antibodies), etc.

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 naturally occurring polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene (polyalkenylene) or polyoxyalkylene polymers or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides. Suitable polymers include, for example, ethylene vinyl alcohol copolymers (commonly known under the generic name EVOH or trade name EVAL); polybutylmethacrylate; poly (hydroxyvalerate); poly (L-lactic acid); polycaprolactone; poly (lactide-co-glycolide); poly (hydroxybutyric acid); poly (hydroxybutyrate-co-valerate); polydioxanone; polyorthoesters; polyanhydrides; poly (glycolic acid); poly (D, L-lactic acid); poly (glycolic acid-co-trimethylenecarbonate); polyphosphates; polyphosphonate urethane; poly (amino acids); cyanoacrylate; poly (trimethylenecarbonate); poly (iminocarbonates); co (ether-esters) (e.g., poly (ethylene oxide) -poly (lactic acid) (PEO/PLA) copolymers); polyalkylene oxalates; polyphosphazene; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid; polyurethane; a silicone; a polyester; a polyolefin; polyisobutylene and ethylene-alpha olefin copolymers; acrylic acid polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinyl halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; a polyvinyl ketone; polyvinyl aromatic hydrocarbons such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-ethylene acetate copolymer; polyamides such as nylon 66 and polycaprolactam; alkyd resin; a polycarbonate; polyoxymethylene; polyimide; polyether; an epoxy resin; polyurethane; a rayon; triacetic acid rayon; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; glass paper; nitrocellulose; cellulose propionate; cellulose ether; amorphous Teflon; poly (ethylene glycol) and carboxymethyl cellulose.

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

Suitable polymers may have an average molecular weight in the range 500Da to 50000Da, for example from 5000Da to 40000Da, or from 25000 to 40000Da. For example, in some embodiments, wherein the subject antibody comprises a poly (ethylene glycol) (PEG) or methoxypoly (ethylene glycol) polymer, the PEG or methoxypoly (ethylene glycol) polymer may have a molecular weight in the range of from about 0.5 kilodaltons (kDa) to 1kDa, from about 1kDa to 5kDa, from 5kDa to 10kDa, from 10kDa to 25kDa, from 25kDa to 40kDa, or from 40kDa to 60 kDa.

In some embodiments, the subject antibodies are covalently linked to PEG polymers. In some embodiments, the subject scFv multimer is covalently linked to the PEG polymer. PEG suitable for conjugation to proteins is typically soluble in water at room temperature and has the general formula R (O-CH ₂-CH₂)_n O-R, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and where n is an integer from 1 to 1000.

The subject antibodies may be glycosylated, e.g., the subject antibodies may include covalently linked carbohydrate or polysaccharide moieties. Glycosylation of antibodies is typically either N-linked or O-linked. The addition of glycosylation sites to antibodies is conveniently accomplished by altering the amino acid sequence such that it comprises an N-or O-linked glycosylation site. Similarly, removal of glycosylation sites can be accomplished by amino acid changes within the native glycosylation site of the antibody.

The subject antibodies can be covalently linked to a second moiety (e.g., lipid, polypeptide other than the subject antibody, synthetic polymer, carbohydrate, etc.) using, for example, glutaraldehyde, homobifunctional crosslinking agents, or heterobifunctional crosslinking agents. Glutaraldehyde crosslinks the polypeptide through their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters or homobifunctional sulfhydryl reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction process in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. The amine containing polypeptide is crosslinked with difunctional NHS esters and imide esters. At a mildly alkaline pH, the imidoester reacts only with primary amines to form imidoamides (imidoamide), and the total charge of the crosslinked polypeptide is unaffected. Homobifunctional sulfhydryl reactive crosslinkers include bismaleimide hexane (BMH), 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB), and 1, 4-bis- (3 ',2' -dithiopyridine) propionic acid amidobutane (DPDPB).

The heterobifunctional crosslinking reagent has two or more different reactive moieties (e.g., an amine reactive moiety and a sulfhydryl reactive moiety) and crosslinks with one of the polypeptides via the amine or sulfhydryl reactive moiety and then reacts with the other polypeptide via the unreacted moiety. A variety of heterobifunctional haloacetyl cross-linkers are available, as are pyridyldithio cross-linkers. Carbodiimides are typical examples of heterobifunctional crosslinking reagents for coupling carboxyl groups with amines, which produce amide linkages.

The subject antibodies will in some embodiments include a "radio-opaque" label, such as one that can be readily visualized using, for example, X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodinated, brominated, or barium salts. Other radiopaque materials are also known and include, but are not limited to, organobismuth derivatives, radiopaque polyurethane rubbers, organobismuth (organobismuth) composites, radiopaque barium polymer composites, and the like.

In some embodiments, the subject antibodies include polyamine modifications. The subject antibodies may be modified with naturally occurring or synthetic polyamines. Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1, 3-deaminated propane, norspermidine (norspermidine), spermidine symmetrical (syn-homospermidine), spermine, thermophilic pentamine (caldopentamine), homothermophilic pentamine (homocaldopentamine) and canavanine (canavalmine). Putrescine, spermidine and spermine are particularly useful. Synthetic polyamines include the empirical formula C _XH_YN_Z, which may be cyclic or acyclic, branched or unbranched, hydrocarbon chains of 3 to 12 carbon atoms, further including 1 to 6NR or N (R) ₂ moieties, wherein R is H, (C ₁-C₄) alkyl, phenyl or benzyl. The polyamine may be attached to the antibody using any standard crosslinking method.

Antibody modification method

The anti-Nectin-4 antibody conjugates of the disclosure may include: 1) An Ig heavy chain constant region conjugated to a moiety of interest; and an Ig light chain constant region conjugated to a moiety of interest; 2) An Ig heavy chain constant region conjugated to a moiety of interest; and an Ig light chain constant region not conjugated to a moiety of interest; or 3) Ig heavy chain constant regions not conjugated to a moiety of interest; and an Ig light chain constant region conjugated to a moiety of interest. The subject anti-Nectin-4 antibody conjugate may also include VH and/or VL domains conjugated to the moiety of interest.

In one example, the antibody may be modified to include 2-formylglycine residues, which may act as a chemical handle for attaching the heterologous moiety. For example, the heavy and/or light chain constant regions of anti-Nectin-4 of the present disclosure may be modified to include an amino acid sequence of a sulfatase motif that can be converted to contain 2-formylglycine (fGly) by the action of 2-Formylglycine Generating Enzymes (FGEs). Such sulfatase motifs may also be referred to herein as FGE modification sites. The action of FGE is directed in a sequence-specific manner, wherein FGE acts on sulfatase motifs located within immunoglobulin polypeptides. The moiety of interest is provided as a component of a reactive partner for aldehyde reaction with the fGly residue of the converted aldehyde tag of the labeled Ig polypeptide. Attaching a moiety of interest to the fGly residue of an aldehyde-tagged Ig polypeptide can be accomplished using a wide variety of commercially available reagents. For example, aminooxy, hydrazide or thiosemicarbazide derivatives of many moieties of interest are suitable reactive partners and are readily available or can be generated using standard chemical methods.

As described above, the amino acid sequence of an anti-Nectin-4 antibody may be modified to include a sulfatase motif comprising serine or cysteine residues that can be converted (oxidized) to 2-formylglycine (fGly) by action of Formylglycine Generating Enzymes (FGEs), either in vivo (e.g., when translating aldehyde-tag containing proteins in cells) or in vitro (e.g., by contacting aldehyde-tag containing proteins with FGEs in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

Sulfatase motif

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

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

It should be noted that while aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it will be readily understood that longer aldehyde tags are contemplated and encompassed by the present disclosure and can be found in the compositions and methods of the present disclosure. Thus, the aldehyde tag may comprise a minimal sulfatase motif of 5 or 6 residues, or may be longer and comprise a minimal sulfatase motif, which sulfatase motif may be flanked by additional amino acid residues on the N-and/or C-terminal side of the motif. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.

The aldehyde tag may be present at or near the C-terminus of the Ig heavy chain; for example, an aldehyde tag may be present within 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminal end of a naturally wild-type Ig heavy chain. The aldehyde tag may be present in the CH1 domain of the Ig heavy chain. The aldehyde tag may be present in the CH2 domain of the Ig heavy chain. The aldehyde tag may be present in the CH3 domain of the 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.

In certain embodiments, the sulfatase motif used may be described by the following formula:

x ¹Z¹X²Z²X³Z³ (SEQ ID NO: 102) (I'), wherein

Z ¹ is cysteine or serine (which may also be represented by (C/S));

Z ² is a proline or alanine residue (which may also be represented by (P/A));

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

X ¹ is present or absent and when 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), is typically L, M, V, S or T, more typically L, M, S or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X ¹ is present; and

X ² and X ³ independently can be any amino acid, but are typically aliphatic amino acids, polar uncharged amino acids, or sulfur-containing amino acids (e.g., other than aromatic amino acids or charged amino acids), such as S, T, A, V, G or C; for example S, T, A, V or G. In one example, the aldehyde tag has the formula L (C/S) TPSR (SEQ ID NO: 103), e.g., LCTPSR (SEQ ID NO: 104) or LSTPSR (SEQ ID NO: 105). Thus, the present disclosure provides antibodies comprising aldehyde-labeled Ig heavy and/or aldehyde-labeled Ig light chains, wherein the aldehyde-labeled Ig antibodies comprise Ig constant region amino acid sequences comprising heavy and/or light chains of such sulfatase motifs.

For example, in some embodiments, the amino acid sequence of the anti-Nectin-4 heavy and/or light chain may be modified to provide a sequence of at least 5 amino acids of formula X ¹Z¹X²Z²X³Z³, wherein

Z ¹ is cysteine or serine;

Z ² is a proline or alanine residue;

z ³ is an aliphatic amino acid or a basic amino acid;

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

X ² and X ³ are each independently any amino acid,

Wherein the sequence is located within or near the solvent accessible loop region of the Ig constant region, and wherein the sequence is not at the C-terminus of the Ig heavy chain.

The sulfatase motif is typically selected so as to be able to be converted by a selected FGE (e.g., FGE present in a host cell expressing the aldehyde marker polypeptide or FGE to be contacted with the aldehyde marker polypeptide in a cell-free in vitro method).

For example, when the FGE is a eukaryotic FGE (e.g., mammalian FGE, including human FGE), the sulfatase motif may have the formula:

X¹CX²PX³Z³(I”)

Wherein the method comprises the steps of

X ¹ may be present or absent, and when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), such as L, M, S or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X ¹ is present;

X ² and X ³ independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than 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; and

Z ³ is a basic amino acid (e.g., arginine (R), and 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).

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).

Containing fGly sequences

Generally, FGE used to promote conversion of cysteine or serine in the sulfatase motif of the aldehyde tag of a target polypeptide to fGly is selected based on the sulfatase motif present in the aldehyde tag. FGE may be native to the host cell in which the aldehyde marker polypeptide is expressed, or the host cell may be genetically modified to express a suitable FGE. In some embodiments, it may be desirable to use a sulfatase motif compatible with human FGE and express the aldehyde marker protein in human cells expressing FGE or in host cells (typically mammalian cells) genetically modified to express human FGE. Generally, FGEs suitable for use in generating fGly-modified antibodies may be obtained from naturally occurring sources or synthetically produced. For example, a suitable FGE may be derived from a biological source that naturally produces FGE or is genetically modified to express a recombinant gene encoding FGE. Nucleic acids encoding a number of FGEs are known in the art and readily available.

After action of FGE on the sulfatase motif, Z ¹ is oxidized to form a 2-formylglycine (fGly) residue. Furthermore, following FGE-mediated transformation and reaction with a reactive partner comprising the moiety of interest, the fGly position at Z ¹ in the above formula is covalently bound to the moiety of interest (e.g., a detectable label, a water-soluble polymer, a polypeptide, a drug, an active agent, etc.). Accordingly, the present disclosure provides anti-Nectin-4 antibodies having amino acid sequences modified to include fGly portions.

Serine or cysteine in the sulfatase motif is modified to fGly when FGE acts on the anti-Nectin-4 heavy and/or light chain. Thus, the fGly-containing sulfatase motif may have the formula:

X¹(fGly)X²Z²X³Z³(SEQ ID NO:127)(I”’)

Wherein the method comprises the steps of

FGly is a formylglycine residue;

Z ² is a proline or alanine residue (which may also be represented by (P/A));

Z ³ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically 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 when present may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X ¹ is present; and

X ² and X ³ independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than 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.

As described above, to produce a conjugate, a polypeptide containing fGly residues can be conjugated to a drug or active agent by reaction of fGly with a reactive moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, as described above) attached to a linker of the drug or active agent to produce a sulfatase motif containing fGly'. As used herein, the term fGly' refers to an amino acid residue of a sulfatase motif coupled to a drug or active agent through a linker as described herein. Accordingly, the present disclosure provides anti-Nectin-4 antibody conjugates (also referred to herein as "anti-Nectin-4 conjugates").

In certain embodiments, the anti-Nectin-4 conjugate comprises a fGly' containing sulfatase motif of the formula:

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)

Wherein the method comprises the steps of

FGly' is an amino acid residue coupled to a drug or active agent via a linker as described herein;

Z ² is a proline or alanine residue (which may also be represented by (P/A));

Z ³ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically 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;

In certain embodiments, the sequence of formula (II) is located at the C-terminus of the heavy chain constant region of an anti-Nectin-4 antibody. In some cases, the heavy chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(II)

Wherein the method comprises the steps of

Z ² is a proline or alanine residue (which may also be represented by (P/A));

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

Wherein the sequence is the C-terminal end of amino acid sequence QKSLSLSPGK (SEQ ID NO: 129), and wherein the sequence may include 1, 2, 3,4, 5, or 5 to 10 amino acids that are not present within the constant region of the natural, wild-type heavy Ig chain.

In certain embodiments, the heavy chain constant region comprises sequence SLSLSPGSL (fGly') TPSRGS (SEQ ID NO: 248) located at the C-terminus of the Ig heavy chain, e.g., in place of the native SLSLSPGK (SEQ ID NO: 249) sequence.

In certain embodiments, the heavy chain constant region comprises sequence SPGSL (fGly') TPSRGS (SEQ ID NO: 130) located at the C-terminus of the Ig heavy chain, e.g., in place of the native SPGK (SEQ ID NO: 131) sequence.

In certain embodiments, the amino acid residue (fGly') coupled to the drug or active agent is located in the light chain constant region of an anti-Nectin-4 antibody. In certain embodiments, the light chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(II)

Wherein the method comprises the steps of

Z ² is a proline or alanine residue (which may also be represented by (P/A));

Wherein the sequence is the C-terminal of amino acid sequence KVDNAL (SEQ ID NO: 132) and/or the N-terminal of amino acid sequence QSGNSQ (SEQ ID NO: 133).

In certain embodiments, the light chain constant region comprises the sequence KVDNAL (fGly') TPSRQSGNSQ (SEQ ID NO: 134).

In certain embodiments, the amino acid residue (fGly') coupled to the drug or active agent is located in the heavy chain CH1 region of an anti-Nectin-4 antibody. In certain embodiments, the heavy chain CH1 region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(II)

Wherein the method comprises the steps of

Z ² is a proline or alanine residue (which may also be represented by (P/A));

Wherein the sequence is the C-terminal of amino acid sequence SWNSGA (SEQ ID NO: 135) and/or the N-terminal of amino acid sequence GVHTFP (SEQ ID NO: 136).

In certain embodiments, the heavy chain CH1 region comprises the sequence SWNSGAL (fGly') TPSRGVHTFP (SEQ ID NO: 137).

FIG. 30A depicts a map of positions showing possible modification sites for the production of 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) and shows possible modification sites in 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 AAG 00909) and shows possible modification sites in the Ig heavy chain. Heavy and light chain numbering is based on full length heavy and light chains.

FIG. 30B depicts an alignment of the heavy chain constant regions of the human immunoglobulin of IgG1(SEQ ID NO:89;GenBank P01857.1)、IgG2(SEQ ID NO:90;GenBank P01859.2)、IgG3(SEQ ID NO:91;GenBank P01860.2)、IgG4(SEQ ID NO:92;GenBank AAB59394.1) and IgA (SEQ ID NO:93;GenBank AAAT74070), which shows that modification sites can provide an aldehyde tag in the heavy chain of the immunoglobulin. Heavy and light chain numbering is based on complete heavy and light chains.

In some embodiments, the sulfatase motif is at a position not or in addition to the C-terminus of the Ig polypeptide heavy chain. An isolated aldehyde-labeled 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 located in or adjacent to a surface accessible loop region of the anti-Nectin-4 polypeptide heavy chain constant region.

Exemplary surface accessible loop regions of the IgG1 heavy chain 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).

Exemplary surface accessible loop regions of the IgG2 heavy chain 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)DGVEVHNAK(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).

Exemplary surface accessible loop regions of the IgG3 heavy chain 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).

Exemplary surface accessible loop regions of the IgG4 heavy chain 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).

Exemplary surface accessible 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).

Exemplary surface accessible loop regions of Ig light chains (e.g., human kappa light chains) include: 1) RTVAAP (SEQ ID NO: 234); 2) PPS; 3) Gly (see, e.g., Gly);4)YPREA(SEQ ID NO:235);5)PREA(SEQ ID NO:236);6)DNALQSGN(SEQ ID NO:237);7)TEQDSKDST(SEQ ID NO:238);8)HK;9)HQGLSS(SEQ ID NO:239); and 10 at position 150 of the human kappa light chain sequence depicted in FIG. 8C) RGEC (SEQ ID NO: 240).

Exemplary surface accessible loop regions of Ig lambda 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).

The HC constant region of an anti-Nectin-4 antibody as disclosed herein may be selected from one of the following sequences: CT-marking (aldehyde label-bold)

/>

In SEQ ID NO. 70, the italic residue at the C-terminal end of the heavy chain constant region replaces the lysine residue at the C-terminal end of the standard IgG1 heavy chain. Bold residues (LCTPSR (SEQ ID NO: 104)) in the italic residues constitute the aldehyde tag, where C is converted to fGly residues by FGE after heavy chain expression to produce L (fGly) TPSR (SEQ ID NO: 245). fGly can be modified to fGly 'to produce L (fGly') TPSR (SEQ ID NO: 246). fGly' refers to the amino acid residues of an anti-Nectin-4 antibody conjugated to a moiety of interest (e.g., a drug). The non-bold residues in the italic residues are additional residues that differ from the standard IgG1 heavy chain sequence.

58Q-1 (aldehyde Label-bold and replace "EEM" with "DEL")

61G-1 (aldehyde Label-bold and replace "EEM" with "DEL")

91N-1 (aldehyde Label-bold and replace "EEM" with "DEL")

116E-1 (aldehyde Label-bold and replace "EEM" with "DEL")

/>

58Q-2 (aldehyde label-bold)

61G-2 (aldehyde Label-bold)

91N-2 (aldehyde label-bold)

116E-2 (aldehyde label-bold)

/>

58Q-3 (aldehyde Label-bold and replace "KKV" with "KRV" and "EEM" with "DEL")

61G-3 (aldehyde Label-bold and replace "KKV" with "KRV" and "EEM" with "DEL")

91N-3 (aldehyde Label-bold and replace "KKV" with "KRV" and "EEM" with "DEL")

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

116E-3 (aldehyde Label-bold and replace "KKV" with "KRV" and "EEM" with "DEL")

/>

58Q-4 (aldehyde Label-bold and replace "KKV" with "KRV')

61G-4 (aldehyde Label-bold and replace "KKV" with "KRV')

91N-4 (aldehyde Label-bold and replace "KKV" with "KRV')

116E-4 (aldehyde Label-bold and replace "KKV" with "KRV')

Bold residues (LCTPSR) constitute aldehyde tags in which C is converted to fGly residues by FGE after heavy chain expression. fGly can be converted to fGly'. fGly' refers to the amino acid residues of an anti-Nectin-4 antibody conjugated to a moiety of interest (e.g., a drug).

Pharmaceutical and active agent

In some cases, the anti-Nectin-4 antibodies of the disclosure have a drug or active agent (e.g., W ¹ in the conjugates of formula (I) described herein) covalently linked to the heavy and/or light chain of the antibody. For example, an antibody conjugate of the present disclosure may include a drug or active agent as substituent W ¹, and if present, a second drug or active agent as substituent W ². Any of a number of drugs are suitable for use as a reactive partner for conjugation to an antibody, or may be modified to render them suitable for use as reactive partners for conjugation to an antibody. "drug" includes small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), and the like.

As used herein, "small molecule drug" refers to a compound, such as an organic compound, that exhibits a pharmaceutical activity of interest and that generally has a molecular weight of no greater than about 800Da or no greater than 2000Da, but may encompass molecules up to 5kDa and may be as large as about 10kDa. Small inorganic molecules refer to molecules that do not contain carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

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

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

In certain embodiments, the drug or active agent may be a duocarmycin or analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof. The duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties, such as, but not limited to, the duocarmycin and analogs and derivatives thereof described herein. Examples of drugs found to be useful in the conjugates and compounds described herein include, but are not limited to, a duocarmycin or a duocarmycin derivative, such as duocarmycin a, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like. In some embodiments, the duocarmycin is a duocarmycin analog such as, but not limited to, adoxolone, bizelesin (bizelesin), or carbozelesin (carzelesin).

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

In certain embodiments, the drug or active agent (e.g., W ¹ and/or W ²) in formulas (I) and (II) described herein is camptothecin or an analog or derivative thereof. For example, in some cases, the camptothecin or analog or derivative thereof is a compound of formula (III):

wherein:

R ³¹ and R ³² are each independently 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 in a cyclic manner to form a 5 or 6 membered cycloalkyl or heterocyclyl ring;

R ³³ and R ³⁴ are each independently 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 linked in a cyclic manner to form a 5 or 6 membered cycloalkyl or heterocyclyl ring;

R ³⁵ is selected from the group consisting of 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 selected from OH and OC (O) R ³⁷; and

R ³⁷ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments of formula (III), the linker L ^A in formula (I) or formula (II) is attached to the compound of formula (III) at R ³¹、R³²、R³³、R³⁴、R³⁵ or R ³⁶. In certain embodiments of formula (III), linker L ^B in formula (I) or formula (II) is attached to the compound of formula (III) at R ³¹、R³²、R³³、R³⁴、R³⁵ or R ³⁶.

In certain embodiments, R ³¹ and R ³² are each independently 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 joined in an cyclic manner to form a 5-or 6-membered cycloalkyl or heterocyclyl ring.

In certain embodiments, R ³¹ is selected from the group consisting of 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. In certain embodiments, R ³¹ is hydrogen. In certain embodiments, R ³¹ is halogen (e.g., F, cl, br, I). In certain embodiments, R ³¹ is hydroxy. In certain embodiments, R ³¹ is amino or substituted amino. In certain embodiments, R ³¹ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³¹ is methyl. In certain embodiments, R ³¹ is C ₂ substituted alkyl, such as-CH ₂CH₂NH(CH(CH₃)₂). In certain embodiments, R ³¹ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³¹ is alkynyl or substituted alkynyl. In certain embodiments, R ³¹ is alkoxy or substituted alkoxy. In certain embodiments, R ³¹ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³¹ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³¹ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³¹ is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³² is selected from the group consisting of 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. In certain embodiments, R ³² is hydrogen. In certain embodiments, R ³² is halogen (e.g., F, cl, br, I). In certain embodiments, R ³² is hydroxy. In certain embodiments, R ³² is amino or substituted amino. In certain embodiments, R ³² is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³² is methyl. In certain embodiments, R ³² is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³² is alkynyl or substituted alkynyl. In certain embodiments, R ³² is alkoxy or substituted alkoxy. In certain embodiments, R ³² is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³² is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³² is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³² is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³¹ and R ³² are optionally linked annularly to form a 5 or 6 membered cycloalkyl or heterocyclyl ring. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 5 or 6 membered cycloalkyl. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 5 or 6 membered heterocyclyl. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 5 membered cycloalkyl. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 6 membered cycloalkyl. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 5 membered heterocyclyl. In certain embodiments, R ³¹ and R ³² are cyclic linked to form a 6 membered heterocyclyl.

In certain embodiments, R ³³ and R ³⁴ are each independently 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 joined in an cyclic manner to form a 5-or 6-membered cycloalkyl or heterocyclyl ring.

In certain embodiments, R ³³ is selected from the group consisting of 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. In certain embodiments, R ³³ is hydrogen. In certain embodiments, R ³³ is halogen (e.g., F, cl, br, I). In certain embodiments, R ³³ is hydroxy. In certain embodiments, R ³³ is amino or substituted amino. In certain embodiments, R ³³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³³ is methyl. In certain embodiments, R ³³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³³ is alkynyl or substituted alkynyl. In certain embodiments, R ³³ is alkoxy or substituted alkoxy. In certain embodiments, R ³³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³³ is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³⁴ is selected from the group consisting of 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. In certain embodiments, R ³⁴ is hydrogen. In certain embodiments, R ³⁴ is halogen (e.g., F, cl, br, I). In certain embodiments, R ³⁴ is hydroxy. In certain embodiments, R ³⁴ is amino or substituted amino. In certain embodiments, R ³⁴ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³⁴ is methyl. In certain embodiments, R ³⁴ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³⁴ is alkynyl or substituted alkynyl. In certain embodiments, R ³⁴ is alkoxy or substituted alkoxy. In certain embodiments, R ³⁴ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³⁴ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³⁴ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³⁴ is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³³ and R ³⁴ are optionally linked annularly to form a 5 or 6 membered cycloalkyl or heterocyclyl ring. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 5 or 6 membered cycloalkyl. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 5 or 6 membered heterocyclyl. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 5 membered cycloalkyl. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 6 membered cycloalkyl. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 5 membered heterocyclyl. In certain embodiments, R ³³ and R ³⁴ are cyclic linked to form a 6 membered heterocyclyl.

In certain embodiments, R ³⁵ is selected from the group consisting of 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. In certain embodiments, R ³⁵ is hydrogen. In certain embodiments, R ³⁵ is halogen (e.g., F, cl, br, I). In certain embodiments, R ³⁵ is hydroxy. In certain embodiments, R ³⁵ is amino or substituted amino. In certain embodiments, R ³⁵ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³⁵ is methyl. In certain embodiments, R ³⁵ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³⁵ is alkynyl or substituted alkynyl. In certain embodiments, R ³⁵ is alkoxy or substituted alkoxy. In certain embodiments, R ³⁵ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³⁵ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³⁵ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³⁵ is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ³⁶ is selected from OH and OC (O) R ³⁷. In certain embodiments, R ³⁶ is OH. In certain embodiments, R ³⁶ is OC (O) R ³⁷.

In certain embodiments, R ³⁷ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ³⁷ is hydrogen. In certain embodiments, R ³⁷ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³⁷ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ³⁷ is alkynyl or substituted alkynyl. In certain embodiments, R ³⁷ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ³⁷ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ³⁷ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³⁷ is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, the compound of formula (III) has the structure of formula (IIIa):

in certain embodiments of the compounds of formula (IIIa), R ³³ is as described above.

In certain embodiments of the compounds of formula (IIIa), R ³⁶ is as described above.

In certain embodiments of the compounds of formula (IIIa), R ³³ is OH and linker L ^A or L ^B is attached at R ³⁶. In certain embodiments of the compounds of formula (IIIa), linker L ^A or L ^B is attached at R ³³ and R ³⁶ is OH.

In certain embodiments, the compound of formula (III) has the structure of formula (IIIb):

In certain embodiments of compounds of formula (IIIb), R ^31a is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^31a is hydrogen. In certain embodiments, R ^31a is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ^31a is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ^31a is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ^31a is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ^31a is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl. In certain embodiments, R ^31a is carboxy. In certain embodiments, R ^31a is a carboxy ester. In certain embodiments, R ^31a is acyl. In certain embodiments, R ^31a is sulfonyl.

In certain embodiments of the compounds of formula (IIIb), R ³⁶ is as described above.

In certain embodiments of the compounds 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 ester, acyl, and sulfonyl, and linker L ^A or L ^B is attached at R ³⁶. In certain embodiments of the compounds of formula (IIIb), linker L ^A or L ^B is attached at R ^31a and R ³⁶ is OH.

In certain embodiments, the compound of formula (III) has the structure of formula (IIIc):

In certain embodiments of the compounds of formula (IIIc), R ^31b is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^31b is hydrogen. In certain embodiments, R ^31b is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ^31b is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ^31b is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ^31b is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ^31b is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl. In certain embodiments, R ^31b is carboxy. In certain embodiments, R ^31b is a carboxy ester. In certain embodiments, R ^31b is acyl. In certain embodiments, R ^31b is sulfonyl.

In certain embodiments of the compounds of formula (IIIc), R ³⁶ is as described above.

In certain embodiments of the compounds 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 ester, acyl, and sulfonyl, and linker L ^A or L ^B is attached at R ³⁶. In certain embodiments of the compounds of formula (IIIc), linker L ^A or L ^B is attached at R ^31b and R ³⁶ is OH.

In certain embodiments, the compound of formula (III) has the structure of formula (IIId):

in certain embodiments of compounds of formula (IIId), R ^32a and R ^32b are each independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl.

In certain embodiments of the compounds of formula (IIId), R ^32a is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^32a is hydrogen. In certain embodiments, R ^32a is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ^32a is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ^32a is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ^32a is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ^32a is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl. In certain embodiments, R ^32a is carboxy. In certain embodiments, R ^32a is a carboxy ester. In certain embodiments, R ^32a is acyl. In certain embodiments, R ^32a is sulfonyl.

In certain embodiments of the compounds of formula (IIId), R ^32b is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^32b is hydrogen. In certain embodiments, R ^32b is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ^32b is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ^32b is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ^32b is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ^32b is heterocyclyl or substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl. In certain embodiments, R ^32b is carboxy. In certain embodiments, R ^32b is a carboxy ester. In certain embodiments, R ^32b is acyl. In certain embodiments, R ^32b is sulfonyl.

In certain embodiments of the compounds of formula (IIId), R ³⁶ is as described above.

In certain embodiments of the 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 ester, acyl, and sulfonyl, and linker L ^A or L ^B is attached at R ³⁶. In certain embodiments of the compounds of formula (IIId), linker L ^A or L ^B is attached at R ^32a or R ^32b, and R ³⁶ is OH. In certain embodiments of the compounds of formula (IIId), linker L ^A or L ^B is attached at R ^32a and R ³⁶ is OH. In certain embodiments of the compounds of formula (IIId), linker L ^A or L ^B is attached at R ^32b and R ³⁶ is OH.

In certain embodiments, the drug is selected from the group consisting of cytotoxins, kinase inhibitors, selective estrogen receptor modulators, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides.

For example, a cytotoxin may include any compound that results in cell death (e.g., necrosis or apoptosis) or reduced cell viability.

Kinase inhibitors may include, but are not limited to, A Davor, african, bosutinib, cetuximab, cobratinib, crizotinib, cabazittinib, dactyltinib, dasatinib, emtrictinib, erdasatinib, erlotinib, futanetinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, xylotinib, nilotinib, pazopanib, pipamatinib, lu Suoti, sorafenib, sunitinib, flecaitinib, vandetanib, valrfenib, and the like.

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

Immunostimulants may 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, and the like.

Oligonucleotide drugs include, but are not limited to, fu Mi Weisen, pegatanib, mi Bomei, idenphos, defibrinode, sodium norcinacate, goldison, viterbi, volanesorsen, inotersen, toff (tofersen), tominersen, and the like.

Aptamer drugs include, but are not limited to, pipamatinib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-a12, NOX-E36, and the like.

Cytokines include, but are not limited to Albinterferon Alfa-2B, aldesleukin, ALT-801, anakinra, anserin, fu Teming, balugrastim, bempegaldesleukin, binetrakin, CINTREDEKIN BESUDOTOX, CTCE-0214, dapoxetine alpha, diniinterleukin, du Lale, elmendocin alpha, enfehlamine, epoetin delta, erythropoietin, human interleukin-2, interferon alpha-2 c, interferon alpha-n 1, interferon alpha-n 3, alfacon-1 interferon, interferon beta-1 a, interferon beta-1B, interferon gamma-1B, interferon kappa, interleukin-1 alpha, interleukin 10, interleukin-7 Lespedezastin, li Peifei Grastin, lorukafusp alpha, maxy-G34, methoxypolyethylene glycol-epoetin beta, moraxella, mo Laisi, nartriptyline, olprine interleukin, glyoxylated febuxostin, PEGylated interleukin 10 (Pegilodecakin), PEGylated interferon alpha-2 a, PEGylated interferon alpha-2B, PEGylated interferon beta-1 a, PEGylated interferon lambda-1 a, recombinant CD 40-ligand, ralatin, romidepsin, sargrastim, thrombopoietin, west Mo Baijie monoclonal antibody (Tucotuzumab celmoleukin), viral macrophage inflammatory protein, and the like.

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

As used herein, "peptide drug" refers to polymeric compounds containing amino acids and is intended to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptidomimetics. Peptide drugs may be obtained by chemical synthesis or produced from a source encoding the gene (e.g., a recombinant source). The molecular weight of the peptide drug may be in the range and may be 200Da to 10kDa or greater. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; an anti-angiogenic peptide; peptides that activate B cells; peptides that activate T cells; an antiviral peptide; peptides that inhibit viral fusion; increasing the production of peptides by one or more lymphocyte populations; an antimicrobial peptide; a growth factor; growth hormone releasing factor; a vasoactive peptide; an anti-inflammatory peptide; peptides that regulate glucose metabolism; antithrombotic peptides; an antinociceptive peptide; vasodilator peptides; platelet aggregation inhibitors; an analgesic; etc.

Additional examples of drugs found to be useful in the conjugates and compounds described herein include, but are not limited to, tubulysin M, spinosad, STAT3 inhibitors, a-amanitine, aurora kinase inhibitors, belotecan, and anthracycline.

In some cases, the drug is a toxin, such as a cytotoxin. Ribosome Inactivating Proteins (RIPs) are examples of such cytotoxins, a class of proteins that are ubiquitous in higher plants. Suitable cytotoxins include, but are not limited to, ricin, abrin, diphtheria toxin, pseudomonas exotoxins (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pokeweed Antiviral Protein (PAP), botulinum toxin, curcin, momordica charantia, and bouganin.

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

Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatins and active analogues and derivatives thereof. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof; and the active analogues and derivatives of the sesquialter mycin.

Agents that act to reduce cell proliferation are known in the art and are widely used. Such agents include alkylating agents such as nitrogen mustards (nitrogen mustard), nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, dichloromethyl diethylamine, cyclophosphamide (Cytoxan ^TM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, chlorozomycin, uramustine, nitrogen mustards (chlormethine), ifosfamide, chlorambucil, pipobromine, triamcinolone, triethylthiophosphamide, busulfan, dacarbazine, and temozolomide.

Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytoside (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), fluorouridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), jetstatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5, 8-diazafolic acid (dideazafolate) (PDDF, CB 3717), 5, 8-diazatetrahydrofolate (dideazatetrahydrofolic acid) (DDATHF), folinic acid, fludarabine phosphate, jetstatin, and gemcitabine.

Suitable natural products and derivatives thereof (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, ara-C, paclitaxelDocetaxel/>Deoxidizing and helping to get the meta-type mycin, mitomycin-C, L-asparaginase and azathioprine; bucona; alkaloids such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics such as anthracycline, daunorubicin hydrochloride (daunorubicin, rubicin, secomycin), idarubicin, doxorubicin, epirubicin, morpholine derivatives, and the like; phenoxazine bicyclic peptides, such as actinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides, such as plicamycin (mithramycin); anthracenediones, such as mitoxantrone; aziridine pyrrolo-indole diones (azirinopyrrolo indolediones), such as mitomycin; macrocyclic immunosuppressants such as cyclosporin, FK-506 (tacrolimus, plectane), rapamycin and the like; etc.

Other antiproliferative cytotoxic agents are novibu (navelbene), CPT-11, anastrozole (anastrazole), letrozole (letrazole), capecitabine, raloxifene, cyclophosphamide, ifosfamide, and droloxifene (droloxafine).

Microtubule-affecting agents with antiproliferative activity are also suitable for use, and include, but are not limited to, colchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatoxin 10 (NSC 376128), maytansine (NSC 153858), rhizomycin (NSC 332598), paclitaxelDerivatives, docetaxel/>Thiocolchicine (NSC 361792), tritylcysteine (TRITYL CYSTERIN), vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

Hormonal modulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocortical steroids such as prednisone, dexamethasone, and the like; estrogens and progestogens, such as medroxyprogesterone acetate, estradiol, clomiphene, tamoxifen, and the like; and adrenocortical inhibitors, such as aminoglutethimide; 17 alpha-ethinyl estradiol; diethylstilbestrol, testosterone, fluoxytestosterone, drotaandrosterone propionate, testosterone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, clomestrane, medroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprorelin, flutamide (Drogenil), toremifene (Fareston) andEstrogens stimulate proliferation and differentiation; thus, compounds that bind to estrogen receptors are used to block this activity.

Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; urea, such as hydroxyurea; and hydrazines, such as N-methyl hydrazine; epipodophyllotoxin; topoisomerase inhibitors; methyl benzyl hydrazine; mitoxantrone; folinic acid; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, deoxyspergualin, azasporine (azasporine), leflunomide, mizoribine, azaspirane (SKF 105685); (ZD 1839,4- (3-chloro-4-fluoroanilino) -7-methoxy-6- (3- (4-morpholinyl) propoxy) quinazoline) and the like.

Paclitaxel (taxanes) are suitable for use. "paclitaxel" includes paclitaxel (paclitaxel), as well as any active paclitaxel derivative or prodrug. "paclitaxel" (which is understood herein to include analogs, formulations and derivatives such as, for example, docetaxel, TAXOL ^TM、TAXOTERE^TM (formulation of docetaxel), 10-deacetyl analogs of paclitaxel, and 3 'n-debenzoyl-3' n-T-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. nos. 5,294,637;5,283,253;5,279,949;5,274,137;5,202,448;5,200,534;5,229,529; and EP 590,267), or obtained from various commercial sources including, for example, SIGMA CHEMICAL co, st.red bean, mo. (from spruce) T7402; or T-1912 from spruce (Taxus yannanensis)).

Paclitaxel is understood to refer not only to the commonly chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., TAXOTERE ^TM docetaxel as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term "paclitaxel" are various known derivatives, including hydrophilic derivatives and hydrophobic derivatives. Paclitaxel derivatives include, but are not limited to, galactose and mannose derivatives; piperazine and piperazine derivatives.

Embodiments of the present disclosure include conjugates in which an antibody is conjugated to one or more drug moieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties. The drug moiety may be conjugated to the antibody at one or more sites of the antibody, as described herein. In certain embodiments, the average drug to antibody ratio (DAR) of the conjugate (molar ratio) ranges from 0.1 to 20, or 0.5 to 20, or 1 to 20, such as 1 to 19, or 1 to 18, or 1 to 17, or 1 to 16, or 1 to 15, or 1 to 14, or 1 to 13, or 1 to 12, or 1 to 11, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In certain embodiments, the conjugate has an average DAR of 1 to 10, such as 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugate has an average DAR of 1 to 10. In certain embodiments, the conjugate has an average DAR of 1 to 5, such as 4. In certain embodiments, the conjugate has an average DAR of 5 to 10, such as 8. Mean refers to an arithmetic mean.

The drug to be conjugated to the polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide. When the drug is a peptide drug, the reactive moiety (e.g., aminoxy or hydrazide can be located at the N-terminal region, the N-terminal, the C-terminal region, the C-terminal, or at a position internal to the peptide). For example, examples of methods relate to the synthesis of peptide drugs having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide may be reacted with a compound comprising a carboxylic acid group and an oxy-N-Boc group. For example, the amino group of the peptide is reacted with 3- (2, 5-dioxopyrrolidin-1-yloxy) propionic acid. Other variations of compounds comprising a carboxylic acid group and an oxy-N-protecting group may include a different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising the carboxylic acid group and the oxy-N-protecting group occurs by standard peptide coupling chemistry. Examples of peptide coupling reagents that may be used include, but are not limited to DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethyl phosphate-1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide), EDC (1- (3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluorourea hexafluorophosphate), DPPA (diphenyl azide phosphate), BOP (benzotriazol-1-yloxy tris (dimethylamino) hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium tetrafluoroborate), TSTU (O- (N-succinimidyl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate), HATU (N- [ (dimethylamino) -1-H-1,2, 3-triazol [4,5,6] -pyridin-1-ylmethylene ] - -N-methyl-ammonium hexafluorophosphate N-oxide), BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphinic chloride), pyBOP ((1-H-1, 2, 3-benzotriazol-1-yloxy) -tris (pyrrolidine) phosphonium tetrafluorophosphate), brOP (bromotris (dimethylamino) phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4 (3H) -one), pyBrOP (bromotris (pyrrolidine) phosphonium hexafluorophosphate). As non-limiting examples HOBt and DIC can be used as peptide coupling reagents.

The peptides comprising the N-protecting group are deprotected to expose amino-oxy functionality. For example, deprotection of the N-oxysuccinimide group is performed according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, protective Groups in Organic Chemistry, 3 rd edition, 1999,John Wiley&Sons,NY and Harrison et al. Some deprotection conditions include hydrazine reagent, amino reagent or sodium borohydride. Deprotection of the Boc protective group may occur with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methyl hydrazine, phenyl hydrazine, sodium borohydride, and methylamine. The products and intermediates can be purified by conventional methods, such as HPLC purification.

One of ordinary skill will appreciate that factors such as pH and steric hindrance (e.g., accessibility of the amino acid residue to react with the reactive partner of interest) are important. Changing the reaction conditions to provide optimal conjugation conditions is well known to those of ordinary skill and conventional in the art. When conjugated to a polypeptide present in or on a living cell, the conditions are selected such that they are physiologically compatible. For example, the pH may be temporarily lowered for a period of time sufficient to allow the reaction to occur, but within a time that the cells are tolerant (e.g., about 30 minutes to 1 hour). The physiological conditions for polypeptide modification on the cell surface may be similar to those used in ketone-azide reactions in modifying cells with cell surface azides (see, e.g., U.S.6,570,040).

Small molecule compounds containing or modified to contain an alpha-nucleophilic group that serves as a reactive partner for the compounds or conjugates disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the disclosure. General methods for chemical synthesis schemes and conditions for synthesizing compounds of interest are known in the art (see, e.g., smith and March, march' S ADVANCED Organic Chemistry: reactions, MECHANISMS, and Structure, fifth edition, wiley-Interscience,2001; or Vogel, A Textbook of Practical Organic Chemistry, including Qualitative Organic Analysis, fourth edition, new York: longman, 1978).

Method for producing antibodies

The subject antibodies may be produced by any known method, for example, conventional synthetic methods for protein synthesis; recombinant DNA methods, and the like.

Where the subject antibody is a single chain polypeptide, it may be synthesized using standard chemical peptide synthesis techniques. In the case where the polypeptide is chemically synthesized, the synthesis may be performed via a liquid phase or a solid phase. Solid-phase polypeptide synthesis (SPPS) is an example of a suitable method for the chemical synthesis of the subject antibodies, wherein the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Various forms of SPPS (such as Fmoc and Boc) can be used to synthesize subject antibodies.

Standard recombinant methods can be used for the production of the subject antibodies. For example, nucleic acids encoding the light and heavy chain variable regions (optionally linked to constant regions) are inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain is operably linked to control sequences in an expression vector that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequence may be a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell (e.g., a COS or CHO cell). Once the vector is integrated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the antibody.

Due to the degeneracy of the codons, a variety of nucleic acid sequences may encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence may be produced by de novo solid phase DNA synthesis or by Polymerase Chain Reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide.

Suitable expression vectors are typically replicated in a host organism either as episomes of host chromosomal DNA or as part of both. Typically, the expression vector comprises a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence.

Coli is an example of a prokaryotic host cell that may be used to clone a polynucleotide encoding the subject antibody. Other suitable microbial hosts for use include bacilli (such as bacillus subtilis) and other enterobacteriaceae (such as salmonella, serratia and various pseudomonas species). Other microorganisms (such as yeast) are also useful for expression. Saccharomyces (e.g., saccharomyces cerevisiae) and Pichia are suitable examples of yeast host cells.

In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in vitro cell culture) can also be used to express and produce polypeptides (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the invention. Suitable mammalian host cells include CHO cell lines, various Cos cell lines, heLa cells, myeloma cell lines and transformed B cells or hybridomas. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters and enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papilloma viruses, cytomegaloviruses and the like.

Once synthesized (one of chemical or recombinant), the whole antibodies, their dimers, individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high Performance Liquid Chromatography (HPLC) purification, gel electrophoresis, etc. (see generally, scopes, protein Purification (Springer-Verlag, n.y., (1982)). The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% (or more) pure, e.g., free of contaminants, such as cellular debris, macromolecular subjects other than antibodies, etc.

Composition and method for producing the same

The antibodies and/or antibody conjugates of the disclosure, e.g., ADCs, may be formulated in a variety of different ways. In general, when the conjugate is a polypeptide-drug conjugate, the conjugate is formulated in a manner compatible with the drug conjugated to the polypeptide, the condition to be treated, and the route of administration to be used.

In some embodiments, a pharmaceutical composition is provided that comprises any of the antibodies or conjugates of the disclosure (e.g., ADC) and a pharmaceutically acceptable excipient.

The antibody or antibody-conjugate, e.g., ADC, may be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and may be formulated for any suitable route of administration, e.g., oral, topical, or parenteral administration. When the conjugates are provided as liquid injections (such as in those embodiments where they are administered intravenously or directly into the tissue), the conjugates may be provided as a ready-to-use dosage form or as a reconstitutable storage stable powder or liquid comprising pharmaceutically acceptable carriers and excipients.

The methods for formulating antibodies and/or conjugates can be adapted from those methods that are readily available. For example, the antibody and/or conjugate may be provided in the form of a pharmaceutical composition comprising a therapeutically effective amount of the antibody and/or conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally comprise other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulations are suitable for administration to mammals, such as those suitable for administration to humans.

For example, the present disclosure provides compositions comprising subject antibodies or antibody-conjugates. In addition to the subject antibodies or antibody-conjugates, the subject antibodies or antibody-conjugate compositions may comprise one or more of the following: salts such as NaCl, mgCl ₂、KCl、MgSO₄, and the like; buffers such as Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-Tris [ hydroxymethyl ] methyl-3-aminopropanesulfonic acid (TAPS), and the like; a solubilizing agent; detergents, for example nonionic detergents, such as tween-20 and the like; protease inhibitors; glycerol; etc.

In certain embodiments, the present disclosure provides compositions comprising pharmaceutical compositions comprising subject antibodies and/or antibody-conjugates. Generally, the formulation includes an effective amount of the subject antibody and/or antibody-conjugate. By "effective amount" is meant a dose sufficient to produce the desired result (e.g., a reduction in the number of cancer cells). In some cases, the desired outcome is at least a reduction in malignant symptoms compared to a control.

Formulations

In the subject methods, the subject antibodies and/or antibody-conjugates can be administered to a host using any convenient method capable of producing the desired therapeutic or diagnostic effect. Thus, the antibodies and/or antibody-conjugates can be incorporated into a variety of formulations for therapeutic administration. More specifically, the subject antibodies and/or antibody-conjugates may be formulated into pharmaceutical compositions by combining with suitable, pharmaceutically acceptable carriers or diluents, and may be formulated as solid, semi-solid, liquid, or gaseous forms of preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

In pharmaceutical dosage forms, the subject antibodies and/or antibody-conjugates may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone, or in appropriate combination with other pharmaceutically active compounds, as well as in combination. The following methods and excipients are merely exemplary and in no way limiting.

For oral dosage forms, the subject antibodies and/or antibody-conjugates may be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, for example in combination with conventional additives such as lactose, mannitol, corn starch or potato starch; in combination with a binder such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; in combination with a disintegrating agent such as corn starch, potato starch or sodium carboxymethyl cellulose; in combination with a lubricant such as talc or magnesium stearate; and if desired, diluents, buffers, wetting agents, preservatives and flavouring agents.

The subject antibodies and/or antibody-conjugates can be formulated into dosage forms for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

Pharmaceutical compositions comprising the subject antibodies and/or antibody-conjugates are prepared by mixing antibodies and/or antibody-conjugates of the desired purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and/or tonicity agents. Acceptable carriers, excipients, and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl parahydroxybenzoate, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; a low molecular weight (less than about 10 residues) polypeptide; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methyl glucosamine, galactosamine, and neuraminic acid; and/or nonionic surfactants such as tween, brij Pluronics, triton-X or polyethylene glycol (PEG).

The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, wherein the lyophilized dosage form is reconstituted with a sterile solution prior to administration. The standard procedure for reconstitution of a lyophilized composition is to add back a lot of pure water (usually corresponding to the volume removed during lyophilization); however, solutions comprising antibacterial agents may be used to produce pharmaceutical compositions for parenteral administration.

Exemplary antibody and/or antibody-conjugate concentrations in the subject pharmaceutical compositions may range from about 1mg/mL to about 200mg/mL or about 50mg/mL to about 200mg/mL or about 150mg/mL to about 200mg/mL.

The aqueous formulation of the antibody and/or antibody-conjugate may be prepared in a pH buffered solution, for example, at a pH of from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for pH in this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers and other organic acid buffers. The buffer concentration may be from about 1mM to about 100mM, or about 5mM to about 50mM, depending on, for example, the desired tonicity of the buffer and formulation.

Lyoprotectants may also be added to protect the labile active ingredient (e.g., protein) against unstable conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants may be included in amounts of about 10nM to 500 nM.

In some embodiments, the subject formulation includes the subject antibody and/or antibody-conjugate and one or more agents (e.g., surfactants, buffers, stabilizers, tonicity agents) and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl p-hydroxybenzoate, benzalkonium chloride, and combinations thereof. In other embodiments, preservatives are included in the formulation, for example, at concentrations ranging from about 0.001 to about 2% (w/v).

For example, the subject formulation may be a liquid or lyophilized formulation suitable for parenteral administration, and may include: about 1mg/mL to about 200mg/mL of the subject antibody conjugate; from about 0.001% to about 1% of at least one surfactant; about 1mM to about 100mM buffer; optionally about 10mM to about 500mM stabilizer; and about 5mM to about 305mM tonicity agent; and has a pH of about 4.0 to about 7.0.

As another example, the parenteral formulation of the subject is a liquid or lyophilized formulation comprising: about 1mg/mL to about 200mg/mL of the subject antibody; 0.04% tween 20w/v;20mM L-histidine; and 250mM sucrose; and has a pH of 5.5.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antibody conjugate of the disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications of the subject antibody conjugates may depend on the particular antibody conjugate employed and the effect to be achieved, as well as the pharmacodynamics associated with each antibody conjugate in the host.

The subject antibodies and/or antibody-conjugates can be administered as injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for dissolution in or suspension in a liquid vehicle may also be prepared prior to injection. The dosage form may also be emulsified or the antibody conjugate encapsulated in a liposomal vehicle.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

In some embodiments, the subject antibodies and/or antibody-conjugates are formulated in controlled release formulations. Sustained release dosage forms may be prepared using methods well known in the art. Suitable examples of sustained-release dosage forms include semipermeable matrices of solid hydrophobic polymers containing the antibody conjugate, wherein the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and L-ethyl glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic-glycolic acid copolymers, and poly-D- (-) -3-hydroxybutyric acid. By using appropriate additives, by controlling the water content and by developing specific polymer matrix compositions, possible loss of biological activity and possible variation of immunogenicity of antibodies comprised in the sustained release dosage form can be prevented.

Physical systems include, but are not limited to, storage systems with rate controlling membranes, such as microcapsules, macrocapsules, and membrane systems; storage systems without rate controlling membranes such as hollow fibers, ultramicroporous cellulose triacetate, and porous polymeric substrates and foams; monolithic systems (monolithic system), including those systems that are physically dissolved in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, environmental agent-invading, and degradable), and materials that are physically dispersed in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, environmental agent-invading, and degradable); a layered structure comprising a storage layer chemically similar to or different from the external control layer; and other physical methods such as osmotic pumps or adsorption onto ion exchange resins.

Chemical systems include, but are not limited to, chemical attack of the polymer matrix (e.g., heterogeneous or homogeneous attack), or bioerosion of the polymer matrix (e.g., heterogeneous or homogeneous).

Dosage of

The appropriate dosage may be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical arts, the dosage of any one patient depends on many factors, including the size of the patient, body surface area, age, the particular compound to be administered, the sex of the patient, time and route of administration, general health, and other drugs to be administered simultaneously. The subject antibodies and/or antibody-conjugates may be administered in an amount of between 1ng/kg body weight and 20mg/kg body weight, e.g., between 0.1mg/kg body weight and 10mg/kg body weight, e.g., between 0.5mg/kg body weight and 5mg/kg body weight, per dose; however, dosages below or above this exemplary range are contemplated, particularly in view of the factors described above. If the regimen is a continuous infusion, it may also be in the range of 1 μg to 10mg per kg body weight per minute.

Those of skill in the art will readily appreciate that the dosage level may vary depending on the function of the particular antibody and/or antibody-conjugate, the severity of the symptoms, and the susceptibility of the subject to side effects. The preferred dosage of a given compound can be readily determined by one of skill in the art by a variety of methods.

Route of administration

The subject antibodies and/or antibody-conjugates are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and topical routes of administration.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral and other enteral and parenteral routes of administration. The route of administration may be combined or adjusted, if desired, depending on the antibody conjugate and/or desired effect. The subject antibodies and/or antibody-conjugate compositions may be administered in a single dose or in multiple doses. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered orally. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered via the inhaled route. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered intranasally. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered topically. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered intracranially. In some embodiments, the subject antibodies and/or antibody-conjugate compositions are administered intravenously.

The antibodies and/or antibody-conjugates can be administered to a host using any available conventional methods and routes suitable for delivering conventional drugs, including systemic or local routes. Generally, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

Parenteral routes of administration other than inhalation include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrahepatic, and intravenous routes, such as any route of administration other than through the digestive tract. Parenteral administration may be performed to achieve systemic or local delivery of the subject antibodies. Where systemic delivery is required, administration typically involves topical or mucosal administration of invasive or systemic absorption of the pharmaceutical dosage form.

The subject antibodies and/or antibody-conjugates may also be delivered to a subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using suppositories) delivery.

By treatment is meant at least amelioration of symptoms associated with a pathological condition afflicting the host, wherein amelioration is used in a broad sense to refer to at least a reduction in the magnitude of parameters (e.g., symptoms) associated with the pathological condition being treated, such as breast cancer, pancreatic cancer, or lung cancer. Thus, treatment also includes situations in which the pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring, or stopped, e.g., terminated) such that the host is no longer suffering from the pathological condition, or at least symptoms that characterize the pathological condition.

In some embodiments, the subject antibodies and/or antibody-conjugates are administered by injection, e.g., for systemic delivery (e.g., intravenous infusion) or to a local site.

Various hosts (where the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") are treatable according to the subject methods. Typically, such hosts are "mammals (mammal)" or "mammals (mammalian)", where these terms are used broadly to describe organisms in the mammalian class, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats) and primates (e.g., humans, chimpanzees, and monkeys). In some implementations, the host will be a person.

Therapeutic method

The present disclosure provides methods of treating diseases or disorders associated with or caused by Nectin-4 positive cells (e.g., cancerous Nectin-4 positive cells or autoreactive Nectin-4 positive cells).

Treating malignant tumor

The present disclosure provides methods of treating malignancies, including solid tumors or hematological malignancies, which generally involve administering an effective amount of a subject antibody and/or antibody-conjugate alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy) to an individual in need thereof (e.g., an individual with a malignancy).

Malignant tumors include, for example, HCC, non-hodgkin's lymphoma, burkitt's lymphoma, multiple myeloma, chronic lymphocytic leukemia, hairy cell leukemia, pre-lymphocytic leukemia, anal cancer, appendiceal cancer, cholangiocarcinoma (i.e., hepatobiliary tract cancer), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, primary unknown Cancer (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenteropathy cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, vulval cancer, and the like.

In some embodiments, an effective amount of the subject antibody and/or antibody-conjugate is an amount effective to reduce the number of cancer cells in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more in one or more doses when administered alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy) as compared to the number of cancer cells in an individual not treated with the antibody conjugate.

In some cases, the cancer is a solid tumor, such as ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

Aspects of the disclosure include methods of delivering a drug to a target site within a subject. The method comprises administering to a subject a pharmaceutical composition comprising a conjugate according to the present disclosure, wherein administration is effective to release a therapeutically effective amount of the drug from the conjugate at a target site in the subject.

In some embodiments, multiple doses of ADC are administered. The frequency of administration of the ADC may vary depending on any of a variety of factors such as the severity of the symptoms, the condition of the subject, and the like. For example, in some embodiments, the ADC is administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, five times a week, six times a week, every other day, every day (qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc.

Combination therapy

In some embodiments, the subject methods of treating a malignancy comprise administering a subject antibody and/or antibody-conjugate and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, cancer chemotherapeutic agents (as described above).

In some embodiments, the method of treatment may comprise administering to the subject a therapeutically effective amount of an immunomodulatory therapeutic. The immunomodulatory therapeutic may be an immune checkpoint inhibitor or an interleukin. The immune checkpoint inhibitor can inhibit A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT or VISTA. An immune checkpoint inhibitor that inhibits PD-1 signaling may be an anti-PD-1 antibody. The anti-PD-1 antibody may be nivolumab, pembrolizumab, alemtuzumab, dewaruzumab, or avilamab. An immune checkpoint inhibitor that inhibits CTLA-4 can be an anti-CTLA-4 antibody. The anti-CTLA-4 antibody can be ipilimumab.

Subject suitable for treatment

A variety of subjects are suitable for treatment with the subject methods. Suitable subjects include any individual, such as a human, suffering from a malignancy; any individual diagnosed with a person with a malignancy, such as a human; any individual, such as a human, who has had a malignancy and is at risk of malignancy recurrence; any individual, e.g., a human, that has been treated for malignancy (e.g., has been treated with a cancer chemotherapeutic agent) with an agent other than the subject anti-Nectin-4 antibody conjugate and that has not responded to the agent; or any individual, such as a human, who has been treated for malignancy with an agent other than the subject anti-Nectin-4 antibody conjugate (e.g., has been treated with a cancer chemotherapeutic agent) and who has initially responded to the agent but subsequently stopped responding (e.g., relapsed). The subject may have a solid tumor, such as ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

Description of the embodiments

Certain embodiments of the present disclosure are described in the items listed below. These embodiments are merely illustrative and are not intended to limit the scope.

1. A conjugate of formula (I):

wherein:

l ^A is a first linker;

L ^B is a second linker;

W ¹ is a first drug;

w ² is a second drug; and

W ³ is an anti-Nectin-4 antibody.

2. The conjugate of clause 1, wherein Z ¹ is CR ⁴.

3. The conjugate of clause 1, wherein Z ¹ is N.

4. The conjugate of clause 1, wherein Z ³ is CR ⁴.

5. The conjugate of clause 1, wherein Z ³ is C-L ^B-W².

6. The conjugate of any one of clauses 1-5, wherein L ^A comprises:

-(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e-(T⁶-V⁶)_f-,

Wherein the method comprises the steps of

A. b, c, d, e and f are each independently 0 or 1;

T ¹、T²、T³、T⁴、T⁵ and T ⁶ are each independently selected from the group consisting of covalent bonds, (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol and AA is an amino acid residue or amino acid analogue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12;

V ¹、V²、V³、V⁴、V⁵ and V ⁶ are each independently selected from the group consisting of: covalent bond 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein each q is an integer from 1 to 6;

7. The conjugate of clause 6, wherein T ¹、T²、T³、T⁴、T⁵ and T ⁶ are each optionally substituted with a glycoside.

8. The conjugate of clause 6, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

9. The conjugate of any one of clauses 7-8, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

10. The conjugate of any one of items 6 to 9,

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CO-;

T ² is an amino acid analog and V ² is-NH-;

T ³ is (PEG) _n and V ³ is-CO-;

T ⁴ is AA and V ⁴ is absent;

T ⁵ is PABC and V ⁵ is absent; and

F is 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is (PEG) _n and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is substituted (C ₁-C₁₂) alkyl and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0.

11. The conjugate of any one of clauses 1-10, wherein L ^B comprises:

-(T⁷-V⁷)_g-(T⁸-V⁸)_h-(T⁹-V⁹)_i-(T¹⁰-V¹⁰)_j-(T¹¹-V¹¹)_k-(T¹²-V¹²)_l-(T¹³-V¹³)_m-,

Wherein the method comprises the steps of

G. h, i, j, k, 1 and m are each independently 0 or 1;

T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each independently selected from the group consisting of covalent bonds, (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide, and ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol and AA is an amino acid residue or amino acid analogue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12;

V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ are each independently selected from the group consisting of: covalent bond 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein each q is an integer from 1 to 6;

12. The conjugate of clause 11, wherein T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each optionally substituted with a glycoside.

13. The conjugate of clause 11, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

14. The conjugate of any one of clauses 12-13, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

15. The conjugate of any one of items 11 to 14,

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is (PEG) _n and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent; and

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is substituted (C ₁-C₁₂) alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent;

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0.

16. The conjugate of any one of clauses 1-15, wherein the conjugate is selected from the group consisting of:

17. the conjugate of any one of clauses 1 to 16, wherein the anti-Nectin-4 antibody is an IgG1 antibody.

18. The conjugate of clause 17, wherein the anti-Nectin-4 antibody is an IgG1 kappa antibody.

19. The conjugate of any one of clauses 1 to 18, wherein the anti-Nectin-4 antibody comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)，

Wherein the method comprises the steps of

X ¹ is present or absent and, when present, can be any amino acid, provided that when the sequence is located at the N-terminus of the conjugate, X ¹ is present;

fGly' is an amino acid residue coupled to a first drug or a second drug, respectively, via a first linker or a second linker;

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue; and

Z ³ is a basic amino acid or an aliphatic amino acid.

20. The conjugate of clause 19, wherein the sequence is L (fGly') TPSR (SEQ ID NO: 246).

21. The conjugate of item 19, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

22. The conjugate of any one of clauses 19 to 21, wherein the sequence is located at the C-terminus of the heavy chain constant region of the anti-Nectin-4 antibody.

23. The conjugate of item 22, wherein the heavy chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128) (II)，

Wherein the method comprises the steps of

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

Wherein the sequence is the C-terminal end of the amino acid sequence SLSLSPG (SEQ ID NO: 247).

24. The conjugate of clause 23, wherein the heavy chain constant region comprises the sequence SPGSL (fGly') TPSRGS (SEQ ID NO: 130).

25. The conjugate of item 23, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

26. The conjugate of any one of clauses 22 to 25, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in the sequence selected from SEQ ID NO:70 and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

27. The conjugate of any one of clauses 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 71, 75, 79 and 83, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

28. The conjugate of any one of clauses 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 72, 76, 80, and 84, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

29. The conjugate of any one of clauses 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 73, 77, 81, and 85, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

30. The conjugate of any one of clauses 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74, 78, 82 and 86, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

31. The conjugate of any one of clauses 19 to 21, wherein the fGly' residue is located in the light chain constant region of the anti-Nectin-4 antibody.

32. The conjugate of clause 31, wherein the light chain constant region comprises the sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128) (II)，

Wherein the method comprises the steps of

fGly' is an amino acid residue coupled to a drug via a linker;

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

Wherein the sequence is the C-terminal of amino acid sequence KVDNAL (SEQ ID NO: 132) and/or the N-terminal of sequence QSGNSQ (SEQ ID NO: 133).

33. The conjugate of clause 32, wherein the light chain constant region comprises the sequence KVDNAL (fGly') TPSRQSGNSQ (SEQ ID NO: 134).

34. The conjugate of item 33, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

35. The conjugate of any one of clauses 19 to 21, wherein the fGly' residue is located in the heavy chain CH1 region of the anti-Nectin-4 antibody.

36. The conjugate of clause 35, wherein the light chain constant region comprises the sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128) (II)，

Wherein the method comprises the steps of

fGly' is an amino acid residue coupled to a drug via a linker;

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

37. The conjugate of clause 36, wherein the heavy chain CH1 region comprises the sequence SWNSGAL (fGly') TPSRGVHTFP (SEQ ID NO: 137).

38. The conjugate of item 29, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

39. The conjugate of any one of clauses 19 to 21, wherein the fGly' residue is located in the heavy chain CH2 region of the anti-Nectin-4 antibody.

40. The conjugate of any one of clauses 19 to 21, wherein the fGly' residue is located in the heavy chain CH3 region of the anti-Nectin-4 antibody.

41. The conjugate of any one of clauses 1 to 40, wherein the anti-Nectin-4 antibody competes with the anti-Nectin-4 antibody for binding to Nectin-4, the anti-Nectin-4 antibody comprising:

42. The conjugate of any one of clauses 1 to 40, wherein the anti-Nectin-4 antibody comprises:

A VH chain comprising a heavy chain CDR 1-3 (HCDR 1-3) of a VH chain having a sequence selected from SEQ ID NOS: 1 to 17; and

A VL chain comprising light chain CDR 1-3 (LCDR 1-3) of a VL chain having a sequence selected from the group consisting of SEQ ID NOs 18 to 31.

43. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

A VH chain comprising a sequence selected from SEQ ID NOs 1 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 31.

44. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

45. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 23.

46. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

47. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 24 to 27.

48. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

A VH chain comprising HCDR 1-3 having a VH chain with a sequence selected from SEQ ID NOs 14 to 17; and

A VL chain comprising LCDR 1-3 of a VL chain having a sequence selected from SEQ ID NOs 28 to 31.

49. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

a VH chain comprising a sequence selected from SEQ ID NOs 14 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 28 to 31.

50. The conjugate of clause 42, wherein the antibody that specifically binds to Nectin-4 comprises:

A VH chain of an anti-Nectin-4 antibody comprising HCDR1-3 having a VH chain selected from the group consisting of SEQ ID NOs 1 to 17 and 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 an amino acid sequence shown by a sequence selected from the group consisting of SEQ ID NOs 1 to 17, wherein any amino acid difference between the VH chain of the anti-Nectin-4 antibody and the sequence selected from the group consisting of SEQ ID NOs 1 to 17 is in a region other than the CDRs; and

A VL chain of an anti-Nectin-4 antibody comprising LCDR 1-3 having a VL chain with a sequence selected from the group consisting of SEQ ID NOs 18 to 31 and comprising an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater or 100% sequence identity to an amino acid sequence shown by a sequence selected from the group consisting of SEQ ID NOs 18 to 31, wherein any amino acid difference between the VL chain of the anti-Nectin-4 antibody and the sequence selected from the group consisting of SEQ ID NOs 18 to 31 is in a region other than a CDR.

51. The conjugate of any one of clauses 42-50, wherein the anti-Nectin-4 antibody comprises: a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs 70 to 86, wherein C in sequence LCTPSR present in the constant region is replaced with fGly.

52. A pharmaceutical composition comprising:

the conjugate of any one of clauses 1 to 51; and

Pharmaceutically acceptable excipients.

53. A method, the method comprising:

administering to a subject an effective amount of the conjugate of any one of clauses 1 to 51 or the pharmaceutical composition of clause 52.

54. A method of treating cancer in a subject, the method comprising:

administering to the subject a therapeutically effective amount of the conjugate of any one of clauses 1 to 51 or the pharmaceutical composition of clause 52, wherein the administration is effective to treat cancer in the subject.

55. The method of clause 54, wherein the cancer is ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

56. The method of clause 55, wherein the cancer is characterized by cancer cells expressing Nectin-4.

57. The method of clause 56, wherein the conjugate binds to Nectin-4.

58. The method of any one of clauses 53 to 57, further comprising administering to the subject a therapeutically effective amount of an immunomodulatory therapeutic.

59. The method of clause 58, wherein the immunomodulatory therapeutic is an immune checkpoint inhibitor or an interleukin.

60. The method of clause 59, wherein the immune checkpoint inhibitor inhibits A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT, and VISTA.

61. The method of clause 60, wherein the immune checkpoint inhibitor that inhibits PD-1 signaling is an anti-PD-1 antibody.

62. The method of clause 61, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, atuzumab, dewaruzumab, or avilamab.

63. The method of clause 60, wherein the immune checkpoint inhibitor that inhibits CTLA-4 is an anti-CTLA-4 antibody.

64. The method of clause 63, wherein the anti-CTLA-4 antibody is ipilimumab.

65. A method of delivering a drug to a target site in a subject, the method comprising:

Administering the conjugate of any one of clauses 1 to 51 or the pharmaceutical composition of clause 52 to a subject, wherein the administering is effective to release a therapeutically effective amount of the drug from the conjugate at a target site of the subject.

66. An anti-Nectin-4 antibody comprising:

67. The anti-Nectin-4 antibody of clause 66, comprising:

a VH chain comprising a sequence selected from SEQ ID NOs 1 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 31.

68. The anti-Nectin-4 antibody of clause 66, comprising:

A VH chain comprising HCDR 1-3 having a VH chain with a sequence selected from SEQ ID NOs 1 to 6;

69. The anti-Nectin-4 antibody of clause 66, comprising:

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

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 23.

70. The anti-Nectin-4 antibody of clause 66, comprising:

71. The anti-Nectin-4 antibody of clause 66, comprising:

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

A VL chain comprising a sequence selected from SEQ ID NOs 24 to 27.

72. The anti-Nectin-4 antibody of clause 66, comprising:

73. The anti-Nectin-4 antibody of clause 66, comprising:

a VH chain comprising a sequence selected from SEQ ID NOs 14 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 28 to 31.

74. The anti-Nectin-4 antibody of clause 66, comprising:

A VH chain of an anti-Nectin-4 antibody comprising HCDR 1-3 having a VH chain selected from the group consisting of SEQ ID NOs 1 to 17 and 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 an amino acid sequence shown by a sequence selected from the group consisting of SEQ ID NOs 1 to 17, wherein any amino acid difference between the VH chain of the anti-Nectin-4 antibody and the sequence selected from the group consisting of SEQ ID NOs 1 to 17 is in a region other than the CDRs; and

A VL chain of an anti-Nectin-4 antibody comprising LCDR 1-3 having a VL chain with a sequence selected from the group consisting of SEQ ID NOs 18 to 31 and comprising an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, or 100% sequence identity to an amino acid sequence shown by a sequence selected from the group consisting of SEQ ID NOs 18 to 31, wherein any amino acid difference between the VL chain of the anti-Nectin-4 antibody and the sequence selected from the group consisting of SEQ ID NOs 18 to 31 is in a region other than a CDR.

75. The anti-Nectin-4 antibody of any one of clauses 66-74, comprising: a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs 70 to 86, wherein C in sequence LCTPSR present in the constant region is replaced with fGly.

76. A pharmaceutical composition comprising:

The antibody of any one of clauses 66 to 75; and

Pharmaceutically acceptable excipients.

77. A method, the method comprising:

administering to a subject an effective amount of the antibody of any one of clauses 66 to 75 or the pharmaceutical composition of clause 76.

78. A method of treating cancer in a subject, the method comprising:

Administering to the subject a therapeutically effective amount of the antibody of any one of items 66-75 or the pharmaceutical composition of item 76, wherein the administration is effective to treat cancer in the subject.

79. The method of clause 78, wherein the cancer is ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

80. The method of clause 79, wherein the cancer is characterized by cancer cells expressing Nectin-4.

81. The method of clause 80, wherein the conjugate binds to Nectin-4.

82. The method of any one of clauses 78 to 81, further comprising administering to the subject a therapeutically effective amount of an immunomodulatory therapeutic.

83. The method of clause 82, wherein the immunomodulatory therapeutic is an immune checkpoint inhibitor or an interleukin.

84. The method of clause 83, wherein the immune checkpoint inhibitor inhibits A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT, and VISTA.

85. The method of clause 84, wherein the immune checkpoint inhibitor that inhibits PD-1 signaling is an anti-PD-1 antibody.

86. The method of clause 85, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, atuzumab, dewaruzumab, or avilamab.

87. The method of clause 84, wherein the immune checkpoint inhibitor that inhibits CTLA-4 is an anti-CTLA-4 antibody.

88. The method of clause 87, wherein the anti-CTLA-4 antibody is ipilimumab.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is a weighted average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pairs; kb, kilobases; pl, picoliter; s or sec, seconds; min, dividing; h or hr, hr; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotide; m., intramuscular (ground); p., intraperitoneal (ly); s.c., subcutaneously; etc. Unless otherwise indicated, the commercially available reagents mentioned in the examples were used according to the manufacturer's instructions. The source of cells identified in the examples and throughout the specification by ECACC registration number is the european collection of cell cultures (ECACC) in sonzburi, uk. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

General Synthesis procedure

Numerous general references are available that provide well-known chemical synthesis schemes and conditions useful for synthesizing the disclosed compounds (see, e.g., SMITH AND MARCH, march' S ADVANCED Organic Chemistry: reactions, MECHANISMS, and Structure, fifth edition, wiley-Interscience,2001; or Vogel,A Textbook of Practical Organic Chemistry,Including Qualitative Organic Analysis,Fourth Edition,New York:Longman,1978).

The compounds described herein may be purified by any purification scheme known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase may be used, including normal and reverse phases, as well as ion resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., introduction to Modern Liquid Chromatography, second edition, l.r.snyder and j.j.kirkland editions, john Wiley and Sons,1979; and THIN LAYER Chromatography, E.Stahl, springer-Verlag editors, new York,1969.

During any process of preparing the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any molecule of interest. This can be done by standard works such as J.F.W.McOmie, "Protective Groups in Organic Chemistry", plenum Press, london and New York1973, T.W.Greene and P.G.M.Wuts, "Protective Groups in Organic Synthesis", third edition, wiley, new York 1999, "THE PEPTIDES"; volume 3 (editor: E.Gross and J.Meienhofer), ACADEMIC PRESS, london and New York 1981, in "Methoden der organischen Chemie", houben-Weyl, fourth edition, volume 15/l, georg THIEME VERLAG, stuttgart 1974, ,H.-D.Jakubke and H.Jescheit,"Aminosauren,Peptide,Proteine",Verlag Chemie,Weinheim,Deerfield Beach,and Basel 1982, and/or in Jochen Lehmann,"Chemie der Kohlenhydrate:Monosaccharide and Derivate",Georg Thieme Verlag,Stuttgart 1974. The protective groups can be removed at a convenient subsequent stage using methods known in the art.

The subject compounds may be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the following schemes.

Example 1: anti NECTIN-4 monoclonal antibodies

Method and results

Antibody discovery and lead selection methods

The mice were immunized with recombinant human nectin-4-His protein. 1500 clones were screened by ELISA to test for reactivity to antigen. Positive hits were confirmed by rescreening human nectin-4-His, cynomolgus monkey nectin-4-His and human CD22-His proteins to identify clones with strong selective binding to human and cynomolgus monkey nectin-4 proteins. The pilot clones were sequenced and generated via transient transfection as recombinant mouse-human chimeric antibodies (antibodies with human kappa light chain and IgG1 constant regions with two aldehyde tag insertions). The antibodies were conjugated to aldehyde-reactive linkers-loads prior to further analysis.

Humanized antibody production method

The 5D9 clone was selected for humanization. Five heavy and five light chain variant sequences were designed (tables 2-3) and antibody variants were constructed by pairing each of these heavy and light chains in all possible combinations. Human kappa light chain and IgG1 constant regions carrying two aldehyde tag insertions were used as constant regions for variant generation. Antibodies were generated via transient transfection and conjugated to aldehyde-reactive-linker loads prior to further analysis.

Antibody discovery and lead selection results

From the 1500 initial clones screened, 27 passed the rescreening process. The 20 cloned sequences of these were successfully recovered (recovery) and recreated as chimeric antibodies carrying two aldehyde tag insertions. The titer of the pilot clone is shown in table 6. ELISA results for the rescreening of twenty clones (as hybridoma supernatants and recombinant proteins) are shown in Table 6.

TABLE 6 titer of lead antibody clones

ELISA method

TABLE 7 Nectin and NECL ELISA reagent

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Nectin and NECLELISA protocol

1.1 Ug/ml antigen in 100uL of PBS was plated on maxisorp well plates.

Maxisorp plate. Incubate overnight at 4 ℃.

3. Wash 4 times with 0.1% tween PBS.

4. Wells were blocked with 200 μ LPBS casein with shaking at room temperature for 2 hours.

5. Wash 4 times with 0.1% tween PBS.

6. The antibody sample was diluted to the appropriate starting concentration.

7. The mAb was serially diluted 1:3 in PBS starting at 3ug/ml (20 nM) in deep well plates. Mixing for 10-15 times. And (3) injection: 300ug/ml (2. Mu.M) mAb can also be used to test physiological concentrations.

8. 100 Μl of mAb dilution was added to 96-well plates.

9. Incubate with shaking at room temperature for 1 hour.

10. Wash/soak 6 times with 0.1% tween PBS.

11. 100UL 1:15000 diluted goat anti-human HRP was added to the wells (diluted with PBS).

12. Incubate with shaking at room temperature for 0.5 hours.

13. Wash 4 times with 0.1% tween PBS.

14. 100 Μl TMB was added and the plate developed until the top well was deep blue.

15. 100 Μl of 2N H ₂SO₄ was added.

16. Absorbance was read on a microplate reader.

Nectin-4 binding ELISA clone selection results

Twenty clones were tested for binding to human nectin-4 by ELISA (figures 1 and 2 and table 8). From these data, 6 clones were selected as the lead binders: 12E11, 3C12, 5D9, 6C8, 7E8 and 7H10 (sequences shown in tables 2 and 3).

TABLE 8 ELISA of monoclonal antibody clones against Nectin-4

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Nectin-4 binding ELISA humanized variant results

Humanized 5D9 variants (sequences shown in tables 2 and 3) (titres shown in table 9) were generated and tested for binding to human nectin-4 by ELISA (figures 6-9). The binding affinities of the test variants were varied (table 9).

ELISA titres for human variants of Table 9.5D9

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In vitro cytotoxicity assay

The cell lines were plated at a density of 5x 10 ⁴ cells/well in 100 μl of growth medium in 96-well plates (Costar 3610). The following day, cells were treated with 20 μl of test preparations serially diluted in culture. After incubation for 5 days at 37℃at 5% CO ₂, promega CellTiter was used according to manufacturer's recommendationsThe reagent measures viability. The GI50 curve is calculated in GRAPHPAD PRISM, normalized to the load concentration.

Results of in vitro cytotoxicity assays-lead clones

Twenty clones were tested for in vitro potency against HEK293 cells overexpressing human nectin-4 (fig. 10-13 and table 10). From these data, 6 clones were selected as lead ADC candidates: 12E11, 3C12, 5D9, 6C8, 7E8 and 7H10 (sequences shown in tables 1 and 2).

TABLE 10 in vitro chimeric monoclonal antibody potency

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In vitro cytotoxicity assay results-humanized variants

Humanized 5D9 variants (sequences shown in tables 2 and 3) (titers shown in table 11) were generated and tested for in vitro potency against HEK 293 cells overexpressing human nectin-4 (fig. 14, 17, 20, 23 and 26). Variants were also tested for in vitro efficacy against human breast cancer cell lines SK-BR-3 (FIGS. 15, 18, 21, 24 and 27) and MDA-MB-468 (FIGS. 16, 19, 22, 25 and 28) expressing nectin-4. The in vitro potency of the different test variants varied (table 11).

TABLE 11 testing in vitro potency of antibody variants

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Nectin and Necl protein family reactive ELISA results

Six of the leader clones were tested for their reactivity towards proteins homologous to nectin-4, particularly other nectins and necl family members (nectin-1, nectin-2, nectin-3, necl-1, necl-2, necl-3, necl-4 and necl-5).

Most clones showed low binding (fig. 3), except for 12E11, which showed low levels of cross-reactivity against most of the nectin-4 related proteins. Thus, variants of 12E11 (sequences shown in tables 2 and 3) were designed, variants of 12E11 were generated, and the binding of variants of 12E11 to the nectin-4 related protein was retested (FIGS. 4 and 5). Some variants showed lower reactivity to the nectin-4 related protein compared to the parental 12E11 clone.

Nectin-4 species reactivity assessment by flow cytometry-method

Human Embryonic Kidney (HEK) 293 cells overexpressing human, cynomolgus monkey, rat or mouse nectin-4 proteins were generated. Cells were lifted (lifted) with Versene to retain cell surface proteins and resuspended in PBS+2% FBS at 10e 6/mL. 100 μl was added to the flowtube to test 10 ⁶ cells/test. The primary antibody was diluted to 0.1. Mu.g/mL and 10. Mu.L was added for a total of 1. Mu.g/test. Primary antibodies (or ADCs) were incubated with cells for 1 hour on ice. Then, the cells were washed 1 time in 2ml of pbs+2% FBS, and detected by adding a secondary antibody. AF 488-conjugated anti-human antibody from Jackson Immunoresearch +50% glycerol-diluted to 1/5 of the concentration in PBS +2% FBS was used at 1 μl/test and 5 μl was added per tube, diluted according to manufacturer's instructions. The second reagent was incubated with the cells for 30 minutes, then the cells were washed 2 times with pbs+2% FBS and analyzed by flow cytometry on a BD FacsCanto instrument equipped with FACSDiva software.

Nectin-4 species reactivity assessment by flow cytometry-results

The pilot clones were tested for binding to human, cynomolgus monkey, rat or mouse nectin-4 protein expressed on the surface of HEK 293 cells. Enrolment mab is included as a positive control and anti-FITC reactive antibody is included as a negative control. All the pilot clones bound human and cynomolgus monkey proteins at comparable levels and to rat proteins at lower levels (table 12). Little reactivity was observed to the mouse nectin-4 protein, consistent with the fact that antibodies were produced in mice.

Table 12 species cross-reactivity of antibody clones.

Example 2: synthesis of MMAE construct 8

Compounds 1 and 4 were obtained commercially from Shanghai Meidixi and used as such. Monomethyl auristatin A5 (MMAE) was purchased from BroadPharm. All other reagents were obtained from commercial sources and used without purification.

Preparation of (R) -2- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -3-oxo-3- ((2- (2- (3-oxo-3- (perfluorophenoxy) propoxy) ethoxy) ethyl) amino) propane-1-sulfonic acid (3)

Carboxylic acid 1 (1.33 g,1.67 mmol) was mixed with pentafluorophenol 2 (1.23 g,6.68 mmol) in 6.5mL anhydrous DMF. The mixture was treated with one portion of EDCI-HCl (0.64 g,3.34 mmol) at room temperature and stirred for 20h until complete consumption as judged by HPLC analysis. The reaction mixture was purified directly by reverse phase chromatography (C18 column, 0-80% acetonitrile-water (containing 0.05% TFA)). The pure fractions were combined, concentrated under vacuum until cloudy, and lyophilized to give tan powder PFP-ester product 3 (1.40 g,1.46mmol,87% yield). LRMS (ESI) m/z 961.2[ M+H ] ⁺, calculated as C ₄₄H₄₅F₅N₆O₁₁ S m/z 961.3.

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamide) propanamido) -5- ((5S, 8S,11S, 12R) -11- ((S) -sec-butyl) -12- (2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl-3, 6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyltriacetate (6)

In a20 mL glass vial, monomethyl auristatin A5 (720 mg,1.0 mmol), 5mL anhydrous DMF and 0.35mL DIPEA (2.0 mmol) were combined at room temperature. The resulting mixture was stirred and treated with PNP carbonate 4 (1014 mg,1.0 mmol) in solid form in small portions, then HOAt (136 mg,1.0 mmol) was added in one portion at room temperature. The reaction mixture was stirred for 6h until the reaction was judged complete (HPLC). The reaction mixture was poured into 30mL of water and the resulting precipitate was isolated by rotation and collected, washed with 5mL of water and briefly dried under high vacuum to give 1.87g of crude product 6 as a pale yellow solid which was taken out for the next step without purification.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- ((5S, 8S,11S, 12R) -11- ((S) -sec-butyl) -12- (2- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl-3, 6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (7)

A solution of crude compound 6 (1.87 g) in 15mL of THF was cooled to 0deg.C in an ice bath and slowly treated with 1M aqueous lithium hydroxide (3 mL). The reaction mixture was stirred at 0 ℃ for 3 hours, then warmed to ambient temperature, treated with 3mL of 1M aqueous lithium hydroxide solution and diluted with 3mL of methanol. The resulting mixture was stirred at room temperature for 3 hours until hydrolysis was complete (HPLC) and then quenched by addition of 1M aqueous HCl to pH 7. The reaction mixture was then concentrated under reduced pressure and washed with 10mL of MTBE. The aqueous layer was purified by reverse phase chromatography (C18 column, 0-40% acetonitrile-water (0.05% TFA)). The pure product fractions were combined, concentrated under reduced pressure, and lyophilized to give compound 7 (730 mg,0.60mmol, 60% yield over 2 steps) as a white powder. LRMS (ESI) m/z 1229.7[ M+H ] ⁺, calculated as C ₆₁H₉₆N₈O₁₈ m/z 1229.7.

Preparation of (2S, 3S,4S,5r, 6S) -6- (5- ((5S, 8S,11S,12 r) -11- ((S) -sec-butyl) -12- (2- ((S) -2- ((1 r,2 r) -3- (((1S, 2 r) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl-3, 6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) -2- ((2S, 5S,18 r) -22- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18- (sulfomethyl) -10, 13-dioxa-3,6,16,19-tetraazadidodecanyl) phenoxy) -3, 4-triazatetradecyl) -2- ((2S, 5S, 18H-pyrrolo [2,3-b ] pyridin-1-yl) 2-hydroxy-2-carboxylic acid

To a stirred solution of compound 7 (730 mg,0.60 mmol) in 3mL of anhydrous DMA was added a solution of DIPEA (0.21 mL,1.2 mmol) and PFP-ester 3 (575 mg,0.60 mmol) in 2mL of DMA at room temperature followed by HOAt (84 mg,0.60 mmol). The resulting mixture was stirred for 30 minutes until judged complete coupling (HPLC analysis) and then treated directly with 1.2mL piperidine at room temperature. After 15min, the reaction mixture was purified by reverse phase chromatography (C18 column, 0-40% gradient of acetonitrile-water). The pure fractions were combined, concentrated under reduced pressure at room temperature, and lyophilized to give compound 8 (806 mg,0.45mmol,75% yield) as a white fluffy powder. LRMS (ESI) m/z 1783.9[ M+H ] ⁺, calculated as C ₈₄H₁₃₀N₁₄O₂₆ S m/z 1783.9.

Example 3: synthesis of Belotekang constructs 20 and 25

Synthetic intermediates 4 and 9 were purchased from Shanghai midecalci and used as such. Belotekang 15 was purchased from AstaTech. All other reagents were obtained from commercial sources and used without purification.

Scheme 1. Synthesis of intermediate 14.

Preparation of (9H-fluoren-9-yl) methyl 2- ((5-amino-1- (3- (tert-butoxy) -3-oxopropyl) -1H-indol-2-yl) methyl) -1, 2-dimethylhydrazine-1-carboxylate (10)

Nitro compound 9 (116 mg,0.20 mmol) was dissolved in 1mL THF and mixed with a solution of ammonium chloride (85 mg,1.6 mmol) in 0.5mL water and 1mL methanol. The resulting mixture was stirred vigorously at room temperature and treated with zinc powder (104 mg,1.6 mmol) in small portions for 5 minutes. The reaction mixture was stirred for 2 hours, the solid was filtered off, the filtrate was diluted with 20mL of saturated aqueous ammonium chloride and extracted with ethyl acetate (2×25 mL). The organic extract was dried over sodium sulfate and the solvent removed under vacuum to give crude product 10 which was taken out for the next step without purification. LRMS (ESI) m/z 555.3[ M+H ] ⁺, calculated as C ₃₃H₃₈N₄O₄ m/z 555.3.

Preparation of (9H-fluoren-9-yl) methyl 2- ((1- (3- (tert-butoxy) -3-oxopropyl) -5- (4- (tert-butoxy) -4-oxobutanamido) -1H-indol-2-yl) methyl) -1, 2-dimethylhydrazine-1-carboxylate (12)

The crude compound 10 (. About.0.20 mmol) was mixed with 4- (tert-butoxy) -4-oxobutanoic acid 11 (40 mg,0.23 mmol) in 2mL of DMF. DIPEA (0.12 mL,0.6 mmol) was added to the mixture at room temperature followed by a portion of PyAOP (110 mg,0.21 mmol). After 30 minutes, the reaction was quenched by pouring into a saturated aqueous ammonium chloride solution, extracted with ethyl acetate, washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give 120mg (0.17 mmol, 85% yield of 2 steps) of product 12 as a dark oil, which was used further without additional purification. LRMS (ESI) m/z 733.4[ M+Na ] ⁺, calculated as C ₄₁H₅₀N₄O₇ m/z 733.4.

Preparation of 4- ((2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1- (2-carboxyethyl) -1H-indol-5-yl) amino) -4-oxobutanoic acid (13)

Bis-tert-butyl ester compound 12 (120 mg,0.17 mmol) was dissolved in a mixture of 2mL of anhydrous DCM, 2mL of TFA and 0.5mL of triisopropylsilane. The resulting mixture was allowed to stand at room temperature for 4 hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography (C18 column, 0-70% v/v gradient of CH ₃CN/H₂ O (containing 0.05% tfa)) to give 53mg (0.09 mmol,53% yield) of diacid product 13.LRMS (ESI) m/z 599.3[ M+H ] ⁺, calculated as C ₃₃H₃₄N₄O₇ m/z599.2.

Preparation of (9H-fluoren-9-yl) methyl 1, 2-dimethyl-2- ((1- (3-oxo-3- (perfluorophenoxy) propyl) -5- (4-oxo-4- (perfluorophenoxy) butyrylamino) -1H-indol-2-yl) methyl) hydrazine-1-carboxylate (14)

To a mixture of diacid 13 (50 mg,0.084 mmol) and pentafluorophenol (46 mg,0.25 mmol) in 2mL of anhydrous THF was added one portion of DCC (51 mg,0.25 mmol) at room temperature. The resulting mixture was stirred for 16 hours, the solid was filtered off, the filtrate was concentrated and purified by reverse phase chromatography (C18 column, 0-100% v/v gradient CH ₃CN/H₂ O (0.05% TFA)). The product containing fractions were concentrated to about 20mL, poured into 50mL of 10% aqueous citric acid and extracted with ethyl acetate (2 x20 mL), dried over sodium sulfate. The solvent was removed under vacuum to give 67mg (0.072 mmol,86% yield) of the dark viscous oily bis-PFP ester product 14.LRMS (ESI) m/z 953.1[ M+Na ] ⁺, calculated as C ₄₅H₃₂F₁₀N₄O₇ m/z 953.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizinyl [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (16)

To a solution of belotekang 15 (HCl salt, 20mg, 43. Mu. Mol) in 2mL of DMF were added 15uL of DIPEA (86. Mu. Mol) and 6mg of HOAt (43. Mu. Mol). The resulting mixture was treated with PNP carbonate 4 (43 mg, 43. Mu. Mol) at room temperature and stirred for 1 hour, then DMF was removed under vacuum. The residue was dissolved in 1mL of MeOH and treated with 1mL of 1M aqueous LiOH. After 10 minutes, 1mL of 1M aqueous HCl was added to the mixture, followed by 1mL of 0.5M pH 4.7 acetate buffer. The resulting mixture was stirred at room temperature for 30min and purified directly by reverse phase HPLC (C18 column, 0-50% v/v gradient of CH ₃CN/H₂ O (containing 0.05% TFA)). The solvent was removed under vacuum to give 17mg (18. Mu. Mol,43% yield) of compound 16 as a glassy yellow solid. LRMS (ESI): M/z945.4[ M+H ] ⁺, calculated as C ₄₇H₅₆N₆O₁₅ M/z 945.4.

Scheme 2 Synthesis of Branch Beloteprednol etabonate construct 20

Preparation of perfluorophenyl 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oic acid ester (18)

In a dried scintillation vial, 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecane-19-oleic acid (17,4817 mg,1 mmol) and pentafluorophenol (268 mg,2 mmol) were combined in 5mL dry THF. The resulting mixture was treated with one portion of DCC (247 mg,1.2 mmol) at room temperature and the reaction mixture was stirred overnight. The precipitated solid was filtered off, the solvent was removed under vacuum, and the residue was purified by reverse phase chromatography (C18 column, 10-100% v/v gradient CH ₃CN/H₂ O (containing 0.05% tfa)) to give 670mg of PFP ester 18 as a colorless oil (570 mg,0.87mmol,87% yield). LRMS (ESI) m/z 654.2[ M+H ] ⁺, calculated as C ₃₂H₃₂F₅NO₈ m/z 654.2.

(2S, 3S,4S,5R, 6S) -6- (2- ((17S, 20S) -1-amino-17-isopropyl-20-methyl-15, 18-dioxo-3, 6,9, 12-tetraoxa-16, 19-diaza-heneicosane-21-amino) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4',6,7] indolizinyl [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (19)

Compound 16 (262 mg,0.22 mmol) was dissolved in 4mL of DMF. To this solution was added DIPEA (105. Mu.L, 0.66 mmol) and PFP ester 18 (181 mg,0.22 mmol) in 0.5mL of DMF at room temperature, followed by HOAt (38 mg,0.22 mmol). The resulting mixture was allowed to stand at room temperature for one hour, then treated directly with 4mL of triethylamine. The reaction mixture was stirred for 5 hours until Fmoc-deprotection was judged complete by HPLC analysis. The reaction mixture was concentrated in vacuo and purified by reverse phase chromatography (C18 column, 0-50% v/v gradient CH ₃CN/H₂ O (containing 0.05% tfa)) to give 185mg (0.16 mmol,73% yield) of compound 19 as a yellow solid. LRMS (ESI) m/z 1192.5[ M+H ] ⁺, calculated as C ₅₈H₇₇N₇O₂ m/z 1192.5.

(2S,3S,4S,5R,6S)-6-(2-((2S,5S)-25-(5-((2S,5S)-1-((2-

((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4',6,7] indolizinyl [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexadec-26-amino) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) -5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazadipentadecanoyl) -5- ((((2- ((2S) -4-ethyl-4-hydroxy-3, 14-dioxo-3, 3,4,12,14-tetrahydro-1H-pyran-yl), preparation of 6, 7-indolizinyl [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (20)

Compound 19 (23 mg, 19. Mu. Mol) was dissolved in 2mL anhydrous DMA. To this solution were added a portion of DIPEA (10. Mu.L, 57. Mu. Mol) and solid bis-PFP ester 14 (8 mg, 8.6. Mu. Mol) at room temperature, followed by HOAt (2.6 mg, 19. Mu. Mol). The resulting mixture was allowed to stand at room temperature for one hour, and then was directly treated with 17. Mu.L of piperidine (172. Mu. Mol). After 20 min, the reaction mixture was purified by reverse phase preparative HPLC (C18 column, 0-50% v/v gradient of CH ₃CN/H₂ O (containing 0.05% TFA)). The pure fractions were lyophilized to give 5.8mg (2.1. Mu. Mol,24% yield) of compound 20 as yellow powder. LRMS (ESI) m/z 1363.1[ M+2H ] ⁺⁺, calculated as C ₁₃₄H₁₇₄N₁₈O₄₃ m/z 1362.6.

Scheme 3 Synthesis of Branch Beloteprednol etabonate construct 25

Preparation of N ⁶ - (((9H-fluoren-9-yl) methoxy) carbonyl) -N ² - (3- (2- (2-methoxyethoxy) ethoxy) propanols) -L-lysine (23)

To a solution of mPEG 8-acid 21 (100 mg,0.24 mmol) in 2mL of anhydrous DMF was added DIPEA (0.13 mL,0.72 mmol) and HATU (93 mg,0.24 mmol) at room temperature. The resulting mixture was stirred for one hour, then Lys (Fmoc) -OH 22 (89 mg,0.24 mmol) was added to the mixture and stirring was continued for one hour. The reaction mixture was purified directly by reverse phase chromatography HPLC (C18, 0-70% v/v MeCN-H ₂ O (containing 0.05% TFA)) to give 120mg of compound 23 as a colourless oil (0.16 mmol,67% yield). LRMS (ESI) m/z 763.4[ M+H ] ⁺, calculated as C ₃₉H₅₈N₂O₁₃ m/z 763.4.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4-aminobutyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadec-ne-35-amino) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4',6,7] indolizinyl [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (24)

To a solution of carboxylic acid 23 (45 mg, 59. Mu. Mol) in 3mL of anhydrous DMF was added DIPEA (21. Mu.L, 120. Mu. Mol) and HATU (22 mg, 59. Mu. Mol) at room temperature. The resulting mixture was stirred for 20 minutes and mixed with 1mL of DMF of amine 16 (55 mg, 58. Mu. Mol). The reaction mixture was stirred at room temperature for 30 minutes, then piperidine (115 μl,1.2 mmol) was added to the mixture. After 20 min, the reaction mixture was directly purified by reverse phase preparative HPLC (C18, 0-50% v/v MeCN-H ₂ O (containing 0.05% TFA)). Lyophilization of the pure fractions gave 34mg (23. Mu. Mol,40% yield) of compound 24 as yellow powder. LRMS (ESI) m/z 1467.7[ M+H ] ⁺, calculated as C ₇₁H₁₀₂N₈O₂₅ m/z 1467.7.

(2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4- (3- (5- ((S) -28- (((S) -1- (((S) -1- ((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4',), 6,7] Inazinyl [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methyl-1-oxobutan-2-yl) carbamoyl) -26, 34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27, 33-diaza-heptadecane-37-amino) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) propanamido) butyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadecane-35 ] Preparation of amino) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4',6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (25)

To a mixture of compound 24 (34 mg, 23. Mu. Mol) and DIPEA (8. Mu.L, 46. Mu. Mol) in 2mL of DMA was added bis-PFP ester 14 (9.4 mg, 10.5. Mu. Mol) at room temperature followed by HOAt (3 mg, 23. Mu. Mol). The resulting mixture was allowed to stand at room temperature for 30 minutes, and then piperidine (21 μl,0.21 mmol) was added to the mixture at room temperature. After 20 min, the reaction mixture was directly purified by reverse phase preparative HPLC (C18, 0-50% v/v MeCN-H ₂ O (containing 0.05% TFA)). Pure fractions were combined and lyophilized to give compound 25 (23 mg,7 μmol,67% yield) as a yellow solid. LRMS (ESI) m/z 1638.3[ M+H ] ²⁺, calculated as C ₁₆₀H₂₂₄N₂₀O₅₃ m/z 1638.8.

Example 4

Bioconjugation, purification and HPLC analysis

Antibodies (15 mg/mL) bearing one aldehyde tag were conjugated to linker-load at 1.1mM each. The reaction was carried out in 20mM sodium citrate, 50mM NaCl pH 5.5 (20/50 buffer) containing 0.85-2.5% DMA at 37℃for 72h. After conjugation, free drug was removed using 30kD MWCO 0.5mL Amicon spin concentrator. The sample was added to a spin concentrator, centrifuged at 15,000Xg for 7 minutes, then diluted with 450. Mu.L of 20mM sodium citrate, 50mM NaCl pH 5.5 and centrifuged again. This process was repeated 10 times. To determine the DAR of the final product, the ADC was checked by analytical chromatography using either HIC (Tosoh # 14947) or PLRP-RP (AGILENT PL1912-1802 1000A,8um,50x 2.1mm) columns. HIC analysis used mobile phase a:1.5M ammonium sulfate, 25mM sodium phosphate pH 7.0, and mobile phase B:25% isopropyl alcohol, 18.75mM sodium phosphate pH 7.0.PLRP analysis used mobile phase a:0.1% aqueous trifluoroacetic acid, and mobile phase B:0.1% acetonitrile solution of trifluoroacetic acid. Prior to PLRP analysis, samples were denatured by adding 50mM DTT, 4M guanidine hydrochloride (final concentration) and heating at 37 ℃ for 30 minutes. To determine aggregation, the samples were analyzed using analytical size exclusion chromatography (SEC; tosoh # 08541) with a mobile phase of 300mM NaCl, 25mM sodium phosphate (pH 6.8) containing 5% isopropanol.

FIG. 34 DAR of 3.74 was generated with the double labeled Nectin-4 VH4/VL1 antibody conjugated to compound 8, as determined by PLRP.

FIG. 36 DAR of 3.73 was generated with the double labeled Nectin-4 VH4/VL5 antibody conjugated to compound 8, as determined by PLRP.

FIG. 38 DAR to 6.89 was generated with the double labeled Nectin-4 VH4/VL1 antibody conjugated to compound 25, as determined by PLRP.

FIG. 40 DAR of 6.86 was generated with the double labeled Nectin-4 VH4/VL5 antibody conjugated to compound 25, as determined by PLRP.

FIG. 42. Single labeled Nectin-4 VH4/VL1 antibody conjugated to compound 25 produced a DAR of 3.16, as determined by PLRP.

FIG. 44A single labeled Nectin-4 VH4/VL5 antibody conjugated to compound 25 produced a DAR of 3.25 as determined by PLRP.

Example 5: xenograft study

NCI-H1781 xenograft method:

Female BALB/c nude mice (5 per group) were inoculated subcutaneously with 2000 ten thousand NCI-H1781 cells in PBS. Treatment was started when the tumors reached an average of 222mm ³ (first day). Animals were dosed intravenously with either vehicle alone or with a DAR of 6.8 conjugated enrolment mab antibody with two aldehyde tag insertions to RED-674. ADC was given intravenously at 5mg/kg on day 0 and day 7. Animals were monitored twice weekly for body weight and tumor size. Animals were euthanized when tumors reached 2000mm ³ or body weight loss exceeded 15%. NCI-H1781 xenograft results:

Aldehyde-conjugated targeting nectin-4 ADCs with topoisomerase I inhibitor loading showed strong tumor regression, including complete response in two of five animals carrying NCI-H1781 xenografts (fig. 29). By the end of the study (day 35), the mean tumor sizes (. + -. SD) for the vehicle control and ADC treated groups were 971.+ -.237 mm ³ and 10.+ -.10 mm ³, respectively.

FIG. 31 shows a graph of NCI-H1781 xenograft studies with single 2.5 or 7.5mg/kg intravenous doses of the listed anti-nectin-4 ADCs on day 0. VH4/VL1 compound 8 (RED-601) and VH4/VL5 compound 8 both use an internal 91N tag and deliver half the loading dose compared to Padcev. Isotype control ADC had minimal activity.

FIG. 32 shows a graph of NCI-H1781 xenograft studies on day 0 using single 2.5 or 7.5mg/kg intravenous doses of either the listed anti-nectin-4 or isotype control ADCs. Preparation of VH4/VL1 Compound 25 (RED-694) was the DAR4 form using a 91N tag and the DAR8 form using a 91N/116E ditag combination. Padcev (imitation) was included as a comparison. Isotype control compound 25ADC had minimal activity.

FIG. 33 shows a graph of NCI-H1781 xenograft studies on day 0 using single 2.5 or 7.5mg/kg intravenous doses of either the listed anti-nectin-4 or isotype control ADCs. Preparation of VH4/VL5 Compound 25 (RED-694) was the DAR4 form using a 91N tag and the DAR8 form using a 91N/116E ditag combination. Padcev (imitation) was included as a comparison. Isotype control compound 25ADC had minimal activity.

Example 6: toxicity study

Toxicity studies performed showed improved tolerance of the nectin-4CH1/CT aldehyde labeled enrolment monoclonal antibody conjugated to topoisomerase I linker-load (Compound 25) compared to Bei Duoting conjugated enrolment monoclonal antibody.

ADC for multi-dose rat toxicity studies

Connector-load	Antibodies to	DAR	% Monomer(s)
				Compound 25	Heavy chain CH 1/CT-labeled enrolment monoclonal antibody	6.47	96.6
Bei Duoting A	Wild-type enrolment monoclonal antibodies	4.17	96.3

Method of

Multi-dose non-GLP rat toxicology study. Male Sprague-Dawley rats (8-9 weeks old, 5 animals/group at study initiation) were given either intravenously with vehicle alone or with a nectin-4 conjugate made with an antibody carrying the enrolment mab variable region of the rat cross-reactive antibody. The ADCs tested were nectin-4 Bei Duoting ADC (Padcev, a mimetic) and enrolment monoclonal antibody compound 25ADC. 10mg/kg was given weekly for 4 doses (days 1, 8, 15 and 22). Animals were observed four times a week 7 days after the last dose. Body weight was recorded. Blood was collected from all animals for clinical pathology on days 5, 12, 19 and 26 and toxico-kinetic analysis (for all doses) was performed 8 hours after dosing and on days 4 and 7. Clinical observations were made daily. The clinical observation scoring system scale ranges from 0 (normal) to 3 (severe), which is shown in table 13.

Table 13: clinical observation scoring system scale

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Results of multi-dose non-GLP rat toxicology study:

In a multi-dose rat study, the tolerance of enrolment mab ADC conjugated to bepotastine or compound 25 at the CH 1/CT-tag site was compared at the same load/same antibody dose level. Padcev (imitation) ADC is toxic to rats at the administered dose, where one animal dies. Padcev (imitation) other animals in the dosing group showed a variety of clinical observations-most related to fur and skin and clinical pathology readings indicate the effect of ADC on liver and hematopoietic system. In contrast, animals receiving compound 25ADC were very resistant to treatment, did not die, had no clinical observations, and the clinical pathology readings reflected the vehicle control group more closely than the Padcev (mock drug) group.

Fig. 46. Clinical observations of rats repeatedly given cross-reactive nectin-4 ADC. Arrows indicate days of administration. No observation was seen in animals given compound 25 conjugate, whereas clinical observations of bepotastine-administered groups averaged 2.5 on day 17 and eventually led to death of the animals.

Fig. 47. Erythrocyte counts in rats repeatedly dosed with vehicle or ADC.

Fig. 47. Neutrophil count in rats repeatedly dosed with vehicle or ADC.

Figure 49 reticulocyte counts in rats repeatedly administered vehicle or ADC.

Figure 50 lymphocyte counts in rats repeatedly dosed with vehicle or ADC.

Fig. 51 platelet counts in rats repeatedly administered vehicle or ADC.

Example 7: pharmacokinetic sample analysis

Method of

Total antibody and total ADC concentrations were quantified by ELISA as previously described and plotted in figure 54. For total antibodies, the conjugate was captured with anti-human IgG-specific antibodies and detected with HRP-conjugated anti-human Fc-specific antibodies. For total ADC, conjugate was captured with anti-human Fab-specific antibody and detected with mouse anti-maytansine primary antibody, followed by HRP-conjugated anti-mouse IgG-subclass 1-specific secondary antibody. Bound secondary antibodies were detected using an Ultra TMB One-STEP ELISA substrate (Thermo Fisher). After quenching the reaction with sulfuric acid, the signal was read by obtaining absorbance at 450nm on a Molecular DEVICES SPECTRA Max M5 microplate reader equipped with softMax Pro software. Data were analyzed using GRAPHPAD PRISM and Microsoft Excel software.

Results: pharmacokinetic analysis of plasma samples from animals in multi-dose non-GLP rat toxicology study #2 demonstrated dose levels and exposure, and demonstrated improved stability of compound 8 conjugates compared to bepotastine ADC (fig. 54)).

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. A conjugate of formula (I):

wherein:

l ^A is a first linker;

L ^B is a second linker;

W ¹ is a first drug;

w ² is a second drug; and

W ³ is an anti-Nectin-4 antibody.

2. The conjugate of claim 1, wherein Z ¹ is CR ⁴.

3. The conjugate of claim 1, wherein Z ¹ is N.

4. The conjugate of claim 1, wherein Z ³ is CR ⁴.

5. The conjugate of claim 1, wherein Z ³ is C-L ^B-W².

6. The conjugate of any one of claims 1-5, wherein L ^A comprises:

Wherein the method comprises the steps of

A. b, c, d, e and f are each independently 0 or 1;

T ¹、T²、T³、T⁴、T⁵ and T ⁶ are each independently selected from the group consisting of covalent bonds, (C ₁-C₁₂) alkyl, substituted (C ₁-C₁₂) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w、(PEG)_n、(AA)_p、-(CR¹³OH)_x -, 4-amino-piperidine (4 AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide, and ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol and AA is an amino acid residue or amino acid analogue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12;

V ¹、V²、V³、V⁴、V⁵ and V ⁶ are each independently selected from the group consisting of covalent bonds 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein each q is an integer from 1 to 6;

7. The conjugate of claim 6, wherein T ¹、T²、T³、T⁴、T⁵ and T ⁶ are each optionally substituted with a glycoside.

8. The conjugate of claim 6, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

9. The conjugate of any one of claims 7-8, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

10. The conjugate according to any one of claim 6 to 9,

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CO-;

T ² is an amino acid analog and V ² is-NH-;

T ³ is (PEG) _n and V ³ is-CO-;

T ⁴ is AA and V ⁴ is absent;

T ⁵ is PABC and V ⁵ is absent; and

F is 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is (PEG) _n and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0; or (b)

Wherein:

T ¹ is (C ₁-C₁₂) alkyl and V ¹ is-CONH-;

T ² is substituted (C ₁-C₁₂) alkyl and V ² is-CO-;

T ³ is AA and V ³ is absent;

T ⁴ is PABC and V ⁴ is absent; and

E and f are each 0.

11. The conjugate of any one of claims 1-10, wherein L ^B comprises:

-(T⁷-V⁷)_g-(T⁸-V⁸)_h-(T⁹-V⁹)_i-(T¹⁰-V¹⁰)_j-(T¹¹-V¹¹)_k-(T¹²-V¹²)_l-(T¹³-V¹³)_m-, Wherein the method comprises the steps of

G. h, i, j, k, 1 and m are each independently 0 or 1;

V ⁷、V⁸、V⁹、V¹⁰、V¹¹、V¹² and V ¹³ are each independently selected from the group consisting of covalent bonds 、-CO-、-NR¹⁵-、-NR¹⁵(CH₂)_q-、-NR¹⁵(C₆H₄)-、-CONR¹⁵-、-NR¹⁵CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO₂-、-SO₂NR¹⁵-、-NR¹⁵SO₂- and-P (O) OH-, wherein each q is an integer from 1 to 6;

12. The conjugate of claim 11, wherein T ⁷、T⁸、T⁹、T¹⁰、T¹¹、T¹² and T ¹³ are each optionally substituted with a glycoside.

13. The conjugate of claim 11, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

14. The conjugate of any one of claims 12-13, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

15. The conjugate according to any one of claim 11 to 14,

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is (PEG) _n and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent; and

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

t ⁸ is (C ₁-C₁₂) alkyl and V ⁸ is-CONH-;

T ⁹ is substituted (C ₁-C₁₂) alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ is absent;

T ¹¹ is PABC and V ¹¹ is absent; and

L and m are each 0.

16. The conjugate of any one of claims 1-15, wherein the conjugate is selected from the group consisting of:

17. The conjugate of any one of claims 1 to 16, wherein the anti-Nectin-4 antibody is an IgG1 antibody.

18. The conjugate of claim 17, wherein the anti-Nectin-4 antibody is an IgG1 kappa antibody.

19. The conjugate of any one of claims 1 to 18, wherein the anti-Nectin-4 antibody comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)，

Wherein the method comprises the steps of

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue; and

Z ³ is a basic amino acid or an aliphatic amino acid.

20. The conjugate of claim 19, wherein the sequence is L (fGly') TPSR (SEQ ID NO: 246).

21. The conjugate of claim 19, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

22. The conjugate of any one of claims 19 to 21, wherein the sequence is located at the C-terminus of the heavy chain constant region of an anti-Nectin-4 antibody.

23. The conjugate of claim 22, wherein the heavy chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)，

Wherein the method comprises the steps of

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

24. The conjugate of claim 23, wherein the heavy chain constant region comprises the sequence SPGSL (fGly') TPSRGS (SEQ ID NO: 130).

25. The conjugate of claim 23, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

26. The conjugate of any one of claims 22 to 25, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown as the sequence selected from SEQ ID No. 70, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID No. 104).

27. The conjugate of any one of claims 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs 71, 75, 79 and 83, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

28. The conjugate of any one of claims 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs 72, 76, 80 and 84, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

29. The conjugate of any one of claims 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs 73, 77, 81 and 85, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

30. The conjugate of any one of claims 19 to 21, wherein the heavy chain constant region of the anti-Nectin-4 antibody comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs 74, 78, 82 and 86, and comprises a fGly' residue instead of C in sequence LCTPSR (SEQ ID NO: 104).

31. The conjugate of any one of claims 19 to 21, wherein the fGly' residue is located in the light chain constant region of an anti-Nectin-4 antibody.

32. The conjugate of claim 31, wherein the light chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)，

Wherein the method comprises the steps of

fGly' is an amino acid residue coupled to a drug via a linker;

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

Wherein the sequence is the C-terminal end of amino acid sequence KVDNAL (SEQ ID NO: 132) and/or the N-terminal end of sequence QSGNSQ (SE ID NO: 133).

33. The conjugate of claim 32, wherein the light chain constant region comprises the sequence KVDNAL (fGly') TPSRQSGNSQ (SEQ ID NO: 134).

34. The conjugate of claim 33, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

35. The conjugate of any one of claims 19 to 21, wherein the fGly' residue is located in the heavy chain CH1 region of an anti-Nectin-4 antibody.

36. The conjugate of claim 35, wherein the light chain constant region comprises a sequence of formula (II):

X¹(fGly')X²Z²X³Z³(SEQ ID NO:128)(II)，

Wherein the method comprises the steps of

fGly' is an amino acid residue coupled to a drug via a linker;

x ² and X ³ are each independently any amino acid;

Z ² is a proline or alanine residue;

Z ³ is a basic amino acid or an aliphatic amino acid, and

37. The conjugate of claim 36, wherein the heavy chain CH1 region comprises the sequence SWNSGAL (fGly') TPSRGVHTFP (SEQ ID NO: 137).

38. The conjugate of claim 29, wherein

Z ³ is selected from R, K, H, A, G, L, V, I and P;

X ¹ is selected from L, M, S and V; and

X ² and X ³ are each independently selected from S, T, A, V, G and C.

39. The conjugate of any one of claims 19 to 21, wherein the fGly' residue is located in the heavy chain CH2 region of an anti-Nectin-4 antibody.

40. The conjugate of any one of claims 19 to 21, wherein the fGly' residue is located in the heavy chain CH3 region of an anti-Nectin-4 antibody.

41. The conjugate of any one of claims 1 to 40, wherein the anti-Nectin-4 antibody competes with the anti-Nectin-4 antibody for binding to Nectin-4 comprising:

42. The conjugate according to any one of claims 1 to 40, wherein the anti-Nectin-4 antibody comprises:

43. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

a VH chain comprising a sequence selected from SEQ ID NOs 1 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 31.

44. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

45. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 23.

46. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

47. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 24 to 27.

48. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

49. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

a VH chain comprising a sequence selected from SEQ ID NOs 14 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 28 to 31.

50. The conjugate according to claim 42, wherein the antibody that specifically binds to Nectin-4 comprises:

51. The conjugate of any one of claims 42-50, wherein the anti-Nectin-4 antibody comprises: a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs 70 to 86, wherein C in sequence LCTPSR present in the constant region is replaced with fGly.

52. A pharmaceutical composition comprising:

the conjugate according to any one of claims 1 to 51; and

Pharmaceutically acceptable excipients.

53. A method, the method comprising:

administering to a subject an effective amount of the conjugate of any one of claims 1 to 51 or the pharmaceutical composition of claim 52.

54. A method of treating cancer in a subject, the method comprising:

Administering to a subject a therapeutically effective amount of the conjugate of any one of claims 1 to 51 or the pharmaceutical composition of claim 52, wherein administration is effective to treat cancer in the subject.

55. The method of claim 54, wherein the cancer is ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

56. The method of claim 55, wherein the cancer is characterized by cancer cells expressing Nectin-4.

57. The method of claim 56, wherein the conjugate binds to Nectin-4.

58. A method of delivering a drug to a target site in a subject, the method comprising:

Administering the conjugate of any one of claims 1 to 51 or the pharmaceutical composition of claim 52 to a subject, wherein the administration is effective to release a therapeutically effective amount of the drug from the conjugate at a target site in the subject.

59. An anti-Nectin-4 antibody comprising:

60. The anti-Nectin-4 antibody according to claim 59, which comprises:

a VH chain comprising a sequence selected from SEQ ID NOs 1 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 31.

61. The anti-Nectin-4 antibody according to claim 59, which comprises:

62. The anti-Nectin-4 antibody according to claim 59, which comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 18 to 23.

63. The anti-Nectin-4 antibody according to claim 59, which comprises:

64. The anti-Nectin-4 antibody according to claim 59, which comprises:

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

A VL chain comprising a sequence selected from SEQ ID NOs 24 to 27.

65. The anti-Nectin-4 antibody according to claim 59, which comprises:

66. The anti-Nectin-4 antibody according to claim 59, which comprises:

a VH chain comprising a sequence selected from SEQ ID NOs 14 to 17; and

A VL chain comprising a sequence selected from SEQ ID NOs 28 to 31.

67. The anti-Nectin-4 antibody according to claim 59, which comprises:

68. The anti-Nectin-4 antibody of any one of claims 59 to 67, comprising: a heavy chain constant region having the amino acid sequence shown in any one of SEQ ID NOs 70 to 86, wherein C in sequence LCTPSR present in the constant region is replaced with fGly.

69. A pharmaceutical composition comprising:

The antibody of any one of claims 59 to 68; and

Pharmaceutically acceptable excipients.

70. A method, the method comprising:

administering to a subject an effective amount of an antibody of any one of claims 59 to 68 or a pharmaceutical composition of claim 69.

71. A method of treating cancer in a subject, the method comprising:

Administering to a subject a therapeutically effective amount of the antibody of any one of claims 59 to 68 or the pharmaceutical composition of claim 69, wherein administration is effective to treat cancer in the subject.

72. The method of claim 71, wherein the cancer is ovarian cancer, ductal breast cancer, lung adenocarcinoma, and pancreatic cancer.

73. The method of claim 72, wherein the cancer is characterized by cancer cells expressing Nectin-4.

74. The method of claim 73, wherein the conjugate binds to Nectin-4.