Antibody drug conjugates and methods of use thereof

Abstract

The present disclosure provides antibody conjugates (e.g., antibody drug conjugates (ADCs). The present disclosure also encompasses methods of producing such conjugates, as well as methods of using same. Also provided are compositions comprising the ADCs of the present disclosure, including, in some cases, pharmaceutical compositions. In certain aspects, methods of using an ADC are provided that include administering to an individual a therapeutically effective amount of an ADC of the present disclosure.

Description

[Technical field]

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

[0002] Introduction The field of protein-small molecule therapeutic conjugates has evolved significantly, providing several clinically beneficial drugs with the promise of providing more drugs in the coming years. Protein-conjugate therapeutics can provide several advantages, for example, due to specificity, functional diversity, and relatively low off-target activity, resulting in fewer side effects. Chemical modification of proteins can extend these advantages by making them more potent, stable, or diverse.

[0003] Several standard chemical transformations are commonly used to create and engineer post-translational modifications on proteins. Several methods exist by which the side chains of specific amino acids can be selectively modified. For example, carboxylic acid side chains (aspartate and glutamate) can be targeted by initial activation with water-soluble carbodiimide reagents and subsequent reaction with amines. Similarly, lysines can be targeted through the use of activated esters or isothiocyanates, and cysteine ​​thiols can be targeted using maleimides and α-halo-carbonyls.

[0004] One of the major obstacles to the creation of chemically modified protein therapeutics or reagents is the production of proteins in biologically active homogeneous form.The conjugation of drugs or detectable labels to polypeptides is difficult to control, and may result in heterogeneous mixtures of conjugates that differ in the number of drug molecules attached and the position of chemical conjugation.In some cases, it may be desirable to use synthetic organic chemistry tools to control the site of conjugation and / or the drug or detectable label conjugated to polypeptide to direct the precise and selective formation of chemical bonds on polypeptides. Summary of the Invention

[0005] The present disclosure provides antibody conjugates (e.g., antibody drug conjugates (ADCs). The present disclosure also encompasses methods of producing such conjugates, as well as methods of using same. Also provided are compositions comprising the ADCs of the present disclosure, including, in some cases, pharmaceutical compositions. In certain aspects, methods of using an ADC are provided that include administering to an individual a therapeutically effective amount of an ADC of the present disclosure. [Brief description of the drawings]

[0006] [Figure 1] Panel A shows a formylglycine generating enzyme (FGE) recognition sequence inserted at the desired location along the antibody backbone using standard molecular biology techniques. Upon expression, FGE, endogenous to eukaryotic cells, catalyzes the conversion of Cys in the consensus sequence to a formylglycine residue (fGly). Panel B of Figure 1 shows an antibody bearing an aldehyde moiety (2 per antibody) that reacts with a hydrazino-iso-Pictet-Spengler (HIPS) linker and payload to generate a site-specifically conjugated ADC. Panel C of Figure 1 shows the HIPS chemistry, which proceeds through an intermediate hydrazonium ion followed by intramolecular alkylation with a nucleophilic indole to generate a stable C-C bond. [Diagram 2] 1 shows a graph of lymphocyte population in rats 5 days after administration, according to an embodiment of the present disclosure. [Diagram 3] 1 shows a graph of circulating aspartate aminotransferase (AST) levels in rats 5 days after administration according to embodiments of the present disclosure. [Figure 4] 1 shows a graph of circulating alanine aminotransferase (ALT) levels in rats 5 days after administration, according to embodiments of the present disclosure. [Diagram 5] 1 shows a graph of red blood cell counts in rats 5 days after administration according to an embodiment of the present disclosure. [Figure 6] 1 shows a graph of hemoglobin levels in rats 5 days after administration according to an embodiment of the present disclosure. [Figure 7] 1 shows a graph of hematocrit levels in rats 5 days after administration according to an embodiment of the present disclosure. [Figure 8] 1 shows a graph from the first Granta xenograft study with a single dose of ADC on day 7 according to an embodiment of the disclosure. [Figure 9] 1 shows a graph of a second Granta xenograft study with a single 2 mg / kg dose of ADC on day 0 according to an embodiment of the disclosure. Use of internal tags 58Q and 91N resulted in superior efficacy with half the DAR compared to the vedotin conjugate. [Figure 10] 1 shows a graph of circulating neutrophil counts in rats repeatedly administered vehicle or ADC, according to embodiments of the disclosure. [Figure 11] 1 shows a graph of circulating monocyte counts in rats repeatedly administered vehicle or ADC, according to embodiments of the disclosure. [Figure 12] 1 shows a graph of red blood cell counts in rats repeatedly administered vehicle or ADC, according to embodiments of the present disclosure. [Figure 13] 1 shows a graph of hemoglobin levels in rats repeatedly administered vehicle or ADC, according to embodiments of the present disclosure. [Figure 14] 1 shows a graph of hematocrit levels in rats repeatedly administered vehicle or ADC, according to embodiments of the present disclosure. [Figure 15] 1 shows a graph of clinical observations in rats repeatedly administered rat cross-reactive Nectin-4 ADC according to an embodiment of the present disclosure. The arrows indicate the days of administration. No observations were observed in animals administered Compound 5 conjugate, while clinical observations in the Vedotin group averaged 2.5 on day 17, culminating in the death of the animals. [Figure 16A] 1 depicts a site map showing potential modification sites for the generation of aldehyde-tagged Ig polypeptides. The upper sequence is the amino acid sequence of the conserved region of an IgG1 light chain polypeptide (SEQ ID NO: 1) showing the potential modification sites in the Ig light chain, and the lower sequence is the amino acid sequence of the conserved region of an Ig heavy chain polypeptide (SEQ ID NO: 2) (GenBank Accession No. AAG00909) showing the potential modification sites in the Ig heavy chain. The numbering of the heavy and light chains is based on the full-length heavy and light chains. [Figure 16B] 1 depicts an alignment of Homo sapiens immunoglobulin heavy chain constant regions for IgG1 (SEQ ID NO:3, GenBank P01857.1), IgG2 (SEQ ID NO:4, GenBank P01859.2), IgG3 (SEQ ID NO:5, GenBank P01860.2), IgG4 (SEQ ID NO:6, GenBank AAB59394.1), and IgA (SEQ ID NO:7, GenBank AAAT74070), showing modification sites where aldehyde tags may be provided in the immunoglobulin heavy chains. The numbering of the heavy and light chains is based on the total heavy and light chains. [Figure 16C] 1 depicts an alignment of Homo sapiens immunoglobulin heavy chain constant regions for IgG1 (SEQ ID NO:3, GenBank P01857.1), IgG2 (SEQ ID NO:4, GenBank P01859.2), IgG3 (SEQ ID NO:5, GenBank P01860.2), IgG4 (SEQ ID NO:6, GenBank AAB59394.1), and IgA (SEQ ID NO:7, GenBank AAAT74070), showing modification sites where aldehyde tags may be provided in the immunoglobulin heavy chains. The numbering of the heavy and light chains is based on the total heavy and light chains. [Figure 16D]1 depicts an alignment of immunoglobulin light chain constant regions, showing modification sites where an aldehyde tag may be provided in the immunoglobulin light chain. Sequence 1 = Homo sapiens kappa light chain constant region, GenBank CAA75031.1, SEQ ID NO: 8. Sequence 2 = Homo sapiens kappa light chain constant region, GenBank BAC0168.1, SEQ ID NO: 9. Sequence 3 = Homo sapiens lambda light chain constant region, GenBank CAA75033, SEQ ID NO: 10. Sequence 4 = Mus musculus light chain constant region, GenBank AAB09710.1, SEQ ID NO: 11. Sequence 5 = Rattus norvegicus light chain constant region, GenBank AAD10133, SEQ ID NO: 12. [Figure 17] Graph of L-82 xenograft study with listed anti-CD30 ADCs at a single intravenous dose on day 0. VH4 / VL4 Compound 8 (RED-601) uses an internal 91N tag and delivers half the payload dose compared to Adcetris. At 50% ADC dose (1.5 mg / kg) and equivalent dose (3 mg / kg), VH4 / VL4 Compound 8 was equally effective compared to Adcetris, with all treatment groups showing 8 complete responses out of 8 mice / group. VH4 / VL4 antibody alone had minimal activity. [Figure 18] Graph of Karpas299 xenograft study with listed anti-CD30 ADCs at a single intravenous dose on day 0. VH4 / VL4 Compound 8 (RED-601) uses an internal 91N tag and delivers half the payload dose compared to Adcetris. At 50% ADC dose (1.5 mg / kg) and equivalent dose (3 mg / kg), VH4 / VL4 Compound 8 produced 5 / 6 and 6 / 6 complete responses compared to Adcetris, and Adcetris produced 6 / 6 complete responses, albeit at twice the payload amount compared to VH4 / VL4 Compound 8. VH4 / VL4 antibody alone had minimal activity. [Figure 19]Graph of NCI-H1781 xenograft study with the listed anti-Nectin-4 ADCs at a single 2.5 or 7.5 mg / kg intravenous dose on day 0. VH4 / VL1 Compound 8 (RED-601) and VH4 / VL5 Compound 8 both use an internal 91N tag and deliver half the payload dose compared to Padcev. Isotype control ADCs had minimal activity. [Figure 20] 1 shows a toxicokinetic analysis of rat plasma samples from multiple dose non-GLP rat toxicity study #2. The analysis confirms the dose levels and demonstrates improved in vivo stability of the enfortumab compound 5 conjugate relative to the enfortumab vedotin conjugate.

[0007] definition "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, straight-chain and branched-chain hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0008] The term "substituted alkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms in the alkyl chain (excluding the C1 carbon atom) are optionally substituted with -O-, -N-, -S-, -S(O), n- (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl), and other heteroatoms, such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic.

[0009] "Alkylene" preferably has 1 to 6, more preferably 1 to 3, carbon atoms, either linear or branched, and optionally includes -O-, -NR 10 -, NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, by way of example, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), isopropylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0010] "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with a substituent, as described for carbon in the definition of "substituted" below.

[0011] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0012] The terms "alkylaminoalkyl", "alkylaminoalkenyl", and "alkylaminoalkynyl" refer to the group R'NHR"-, where R' is an alkyl group, as defined herein, and R" is an alkylene, alkenylene, or alkynylene group, as defined herein.

[0013] The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0014] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0015] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0016] The term "alkoxyamino" refers to the group -NH-alkoxy, where alkoxy is defined herein.

[0017] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group and includes, by way of example, groups such as trifluoromethoxy.

[0018] The term "haloalkyl" refers to a substituted alkyl group as described above, in which one or more hydrogen atoms on the alkyl group are replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0019] The term "alkylalkoxy" refers to -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0020] The term "alkylthioalkoxy" refers to -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl groups, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0021] "Alkenyl" refers to straight or branched chain hydrocarbyl groups having from 2 to 6 carbon atoms, preferably from 2 to 4 carbon atoms, and having at least 1, and preferably 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0022] The term "substituted alkenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0023] "Alkynyl" refers to straight or branched chain monovalent hydrocarbyl groups having from 2 to 6 carbon atoms, preferably from 2 to 3 carbon atoms, and having at least 1, and preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0024] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0025] "Alkynyloxy" refers to the group -O-alkynyl, where alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0026] "Acyl" means HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, hetero ... "C(O)-," "heterocyclyl-C(O)-," and "substituted heterocyclyl-C(O)-" groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CHC(O)-.

[0027] "Acylamino" is -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) heterocyclic and NR 20 C(O)-substituted heterocyclic group, where R 20is hydrogen or alkyl, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0028] The term "aminocarbonyl" or "aminoacyl" means C(O)NR 21 R 22 R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0029] "Aminocarbonylamino" is -NR 21 C(O)NR 22 R 23 R 21 , R 22 , and R 23 is independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups are joined to form a heterocyclyl group.

[0030] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0031] 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-, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0032] "Aminosulfonyl" is -SONR 21 R 22 R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0033] "Sulfonylamino" is -NR 21 SO2R 22 R 21 and R 22is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 21 and R 22 are optionally joined together with the atoms bound to them to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0034] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as present in a phenyl group) or a ring system having multiple fused rings, which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl). This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition of the aryl substituent, such aryl groups may be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0035] "Aryloxy" refers to the group -O-aryl, where aryl is as defined herein and includes, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups, also as defined herein.

[0036] "Amino" refers to the group -NH2.

[0037] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0038] The term "azido" refers to the group --N3.

[0039] "Carboxyl", "carboxy", or "carboxylate" refers to -COH or a salt thereof.

[0040] The term “carboxyl ester” or “carboxy esters” or “carboxyalkyl” or “carboxylalkyl” refers to —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, — refers to the groups C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0041] "(Carboxyl ester)oxy" or "carbonate" refers to -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cyclo ... "Heteroaryl" refers to the groups -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic, and -OC(O)O-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0042] "Cyano" or "nitrile" refers to the group --CN.

[0043] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having a single cyclic ring or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0044] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, SO2-aryl, and -SO2-heteroaryl.

[0045] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having a single or multiple rings and at least one double bond, preferably 1 to 2 double bonds.

[0046] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having from 1 to 5 substituents, or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0047] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.

[0048] "Cycloalkoxy" refers to -O-cycloalkyl.

[0049] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0050] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0051] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0052] "Heteroaryl" refers to an aromatic group of 1-15 carbon atoms, such as 1-10 carbon atoms in the ring, and 1-10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (such as pyridinyl, imidazolyl, or furyl) or multiple condensed rings (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl) in the ring system, where at least one ring in the ring system is aromatic. To satisfy valency requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent, such as an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of the heteroaryl substituent, such heteroaryl groups may be optionally substituted with from 1 to 5 substituents, or from 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0053] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are as defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0054] "Heteroaryloxy" refers to -O-heteroaryl.

[0055] "Heterocycle", "heterocyclic", "heterocycloalkyl", and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridged ring systems and spiro ring systems, and having 3-20 ring atoms, including 1-10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of a heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. In order to satisfy valency requirements, any heteroatom in such a heterocyclic ring may or may not be bonded to one or more H or one or more substituents, such as alkyl groups or other substituents as described herein.

[0056] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, furan, and the like. These include, but are not limited to, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0057] Unless otherwise constrained by the definition of a heterocyclic substituent, such heterocyclic groups may be optionally substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0058] "Heterocyclyloxy" refers to the group --O-heterocyclyl.

[0059] The term "heterocyclylthio" refers to a heterocyclic -S- group.

[0060] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0061] The term "hydroxyamino" refers to the group -NHOH.

[0062] "Nitro" refers to the -NO2 group.

[0063] "Oxo" refers to the atom (=O).

[0064] "Sulfonyl" refers to SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0065] "Sulfonyloxy" refers to the groups -OSO2-alkyl, OSO2-substituted alkyl, OSO2-alkenyl, OSO2-substituted alkenyl, OSO2-cycloalkyl, OSO2-substituted cycloalkyl, OSO2-cycloalkenyl, OSO2-substituted silcoalkenyl, OSO2-aryl, OSO2-substituted aryl, OSO2-heteroaryl, OSO2-substituted heteroaryl, OSO2-heterocyclic, and OSO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0066] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0067] "Thiol" refers to the group --SH.

[0068] The term "thioxo" or "thioketo" refers to the atom (=S).

[0069] The term "alkylthio" or "thioalkoxy" refers to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, the sulfur can be oxidized to -S(O)-. The sulfoxide can exist as one or more stereoisomers.

[0070] The term "substituted thioalkoxy" refers to the group --S-substituted alkyl.

[0071] The term "thioaryloxy" refers to an aryl-S- group, where aryl is as defined herein and includes optionally substituted aryl groups, as defined herein.

[0072] The term "thioheteroaryloxy" refers to a heteroaryl-S- group, where heteroaryl is as defined herein and includes optionally substituted aryl groups, also as defined herein.

[0073] The term "thioheterocyclooxy" refers to the group heterocyclyl-S-, in which heterocyclyl is as defined herein, and includes optionally substituted heterocyclyl groups, also as defined herein.

[0074] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can mean that one or more hydrogen atoms of the particular group or radical are replaced, each independently of the other, with the same or different substituents defined below.

[0075] In addition to the groups disclosed for each individual term herein, substituents to replace one or more hydrogens on a saturated carbon atom in a particular group or radical (such as =O, =NR, etc., which replace any two hydrogens on a single carbon) are also included. 70 , =N-OR 70 , =N2 or =S) is, unless otherwise specified, -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70)2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -O-C(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; each R 70 are independently hydrogen or R 60 And each R 80 is independently R 70 or alternatively, two R 80together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, which may optionally contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, of which N may have -H or C1-C3 alkyl substitution; + is a counterion with a net single positive charge. + are independently, for example, alkali ions, e.g., K + , Na + , Li + ammonium ions, e.g. + N(R 60 ) 4; or alkaline earth ions, e.g., [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (the subscript 0.5 means that one of the counterions to such divalent alkaline earth ions may be the ionized form of a compound of the invention and the other may be a typical counterion such as chloride, or a two ionized compound disclosed herein may serve as the counterion to such divalent alkaline earth ions, or a doubly ionized compound of the invention may serve as the counterion to such divalent alkaline earth ions). A specific example is -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

[0076] In addition to the disclosure herein, the substituents of hydrogen on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl, and heteroaryl groups are, unless otherwise specified, —R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 , -C(NR 70 )R 70 , and NR 70 , -C(NR 70 )NR 80 R 80 and R60 , R 70 , R 80 , and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + Provided that:

[0077] In addition to the groups disclosed for each individual term herein, the substituents of the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70, -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 、 and M + is as previously defined.

[0078] Further to the disclosure herein, in certain embodiments a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0079] In all of the substituted groups defined above, it is understood that polymers arrived at by defining a substituent that itself has a further substituent (e.g., a substituted aryl having a substituted aryl group as a substituent, which itself is substituted with a substituted aryl group, which in turn is substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0080] Unless otherwise indicated, the naming of substituents not expressly defined herein is arrived at by naming the terminal portion of the functionality before the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0081] For any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0082] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt that contains a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which are derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, as well as, in the case where the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0083] The term "salt thereof" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharma- ceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those in which the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0084] "Solvate" refers to a complex formed by the combination of solvent molecules with molecules or ions of a solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0085] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atomic connectivity but different arrangements of atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0086] "Tautomers" refers to alternative forms of molecules that differ only in the electronic bonding of the atoms and / or in the position of the protons, for example, enol-keto and imine-enamine tautomers, or tautomers of heteroaryl groups containing the -N=C(H)-NH- ring atom arrangement, 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.

[0087] It is to be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include salts, solvates, and all permutations of stereoisomers, e.g., solvates of pharma- ceutically acceptable salts of a stereoisomer of the subject compound.

[0088] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of tetramers, which in turn are composed of two dimers of a heavy and light chain polypeptide), single chain antibodies (e.g., scFv), fragments of antibodies (e.g., whole chain antibodies or single chain antibody fragments) that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies may be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). The antibody may also be bound to a solid support, including, but not limited to, a polystyrene plate or bead. Also encompassed by the term are Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to the antigen, as well as monoclonal antibodies. The antibody may be monovalent or bivalent. An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0089] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) still has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0090] "Fab" fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0091] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0092] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.

[0093] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragments are composed of a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0094] As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents, expressed as a dissociation constant (Kd). The affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1000-fold higher than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or higher. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0095] The term "binding" refers to a direct association between two molecules due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, interactions such as salt bridges and water bridges. The subject antibodies specifically bind to an epitope within a polypeptide, e.g., a human polypeptide, e.g., a glycosylated polypeptide or a fragment thereof. Nonspecific binding is about 10 -7 Binding with an affinity of less than 10 M, e.g. -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0096] The term "specifically binds" in the context of antibodies and antigens means that the antibody specifically binds to an antigen, e.g., at a concentration of about 10 5 M -1 Affinity or K a (i.e., the equilibrium association constant for a particular binding interaction, which has units of 1 / M).

[0097] "High affinity" binding is defined as a binding affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or more than K a Alternatively, affinity may be expressed in units of M (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K D In some embodiments, specific binding can be defined as the binding capacity of an antibody that is greater than or equal to about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10-8 M or less, or about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K below M D The binding affinity of an antibody to an antigen can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, using surface plasmon resonance (SPR) technology (e.g., BIAcore2000 instrument using general procedures outlined by the manufacturer), by radioimmunoassay, etc.

[0098] As used herein, the term "framework" when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions in the variable region of an antibody. Variable region frameworks are generally discontinuous amino acid sequences that are about 100-120 amino acids in length, but are intended to refer to only those amino acids outside the CDRs. As used herein, the term "framework region" is intended to mean each domain of the framework that is separated by the CDRs.

[0099] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region or may comprise a constant region with pre-existing amino acid sequence modifications, such as additions, deletions, and / or substitutions.

[0100] In the context of an Ig polypeptide, the term "constant region" is well understood in the art and refers to the C-terminal region of an Ig heavy chain or an Ig light chain. The Ig heavy chain constant region comprises the CH1, CH2, and CH3 domains (and the CH4 domain, if the heavy chain is a μ or ε heavy chain). In a native Ig heavy chain, the CH1, CH2, CH3 (and CH4, if present) domains start immediately after (C-terminal to) the heavy chain variable (VH) region and are each about 100 to about 130 amino acids in length. In a native Ig light chain, the constant region starts immediately after (C-terminal to) the light chain variable (VL) region and is about 100 to 120 amino acids in length.

[0101] An "epitope" is a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous or non-contiguous amino acids juxtaposed by protein folding (e.g., tertiary folding). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by folding are typically lost on treatment with denaturing solvents. Epitopes typically include at least 3, more commonly at least 5 or 8-10 amino acids in a linear or spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Several commercial laboratories offer epitope mapping services. An epitope bound by an antibody immunoreactive with a membrane-associated antigen may be present on the surface of a cell (e.g., within the extracellular region of a transmembrane protein), such that such epitope is considered cell-surface accessible, solvent accessible, and / or cell-surface exposed.

[0102] "Genetically encodable," as used in reference to an amino acid sequence of a polypeptide, peptide, or protein, means that the amino acid sequence is composed of amino acid residues that can be produced by transcription and translation of a nucleic acid encoding that amino acid sequence, and that transcription and / or translation can occur in a cell or in a cell-free in vitro transcription / translation system.

[0103] 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 that are suitable for prokaryotic systems include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic systems may utilize promoters, polyadenylation signals, and enhancers.

[0104] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate the initiation of translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation or through an amplification reaction. Synthetic oligonucleotide adaptors or linkers can be used to link sequences in accordance with conventional practices.

[0105] The term "expression cassette" as used herein refers to a segment of nucleic acid, usually DNA, that can be inserted into a nucleic acid (e.g., by use of restriction sites compatible with ligation into a construct of interest, or by homologous recombination into a construct of interest or into a host cell genome). Generally, the nucleic acid segment comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. The expression cassette can also include elements that facilitate expression of the polynucleotide encoding the polypeptide of interest in a host cell, e.g., a mammalian host cell. These elements can include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

[0106] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99%, by weight of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment is absent. In some cases, an isolated antibody is prepared by at least one purification step.

[0107] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are produced and paired by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, and serum are examples of tissues that produce natural antibodies. For example, an antibody produced by antibody-producing cells isolated from a first animal immunized with an antigen is a natural antibody.

[0108] 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 region, constant region, or CDR) that are replaced with a correspondingly positioned amino acid from a human antibody. Generally, a humanized antibody generates a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting, veneering or resurfacing, chain shuffling, and the like. In certain embodiments, framework substitutions are identified by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, and to identify unusual framework residues at specific positions. Thus, the antibodies described above can be humanized using methods well known in the art.

[0109] In certain embodiments, the antibody molecules disclosed herein comprise a heavy chain comprising a variable heavy chain region as provided herein and a human IgG1 constant region having the amino acid sequence as set forth in UniProt:P01857-1, version 1. In certain embodiments, the antibody molecules disclosed herein comprise a light chain comprising a variable light chain region as provided herein and a human light chain constant region. In certain embodiments, the human light chain constant region is a human kappa light chain constant region having the amino acids as set forth in UniProtKB / Swiss-Prot:P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibodies may comprise mutations, e.g., substitutions to modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody-dependent cellular cytotoxicity (ADCC). The numbering of substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is commonly used when referring to residues within an immunoglobulin heavy chain constant region (e.g., the EU index as reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). "Kabat-like EU index" refers to the residue numbering of the human IgG1 EU antibody.

[0110] The term "chimeric antibody" refers to an antibody in which the light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody can be joined to human constant segments such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein made up of variable or antigen binding domains from a mouse antibody and constant or effector domains from a human antibody, although domains from other mammalian species can also be used.

[0111] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless specifically indicated otherwise, "polypeptide", "peptide" and "protein" can include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells), immunologically tagged proteins, and the like. In the context of an antibody, it is clear that a chain or domain includes a polypeptide.

[0112] A "native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include a modified amino acid residue.

[0113] The terms "amino acid analog", "unnatural amino acid", and the like are used interchangeably and can include amino acid-like compounds that are similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D forms, as well as L forms of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more natural amino acids, with the difference(s) being one or more modified groups in the molecule. Such modifications can include, but are not limited to, replacement of an atom (such as N) with a related atom (such as S), addition of a group (such as methyl or hydroxyl) or an atom (such as Cl or Br), deletion of a group, replacement of a covalent bond (replacement of a single bond with a double bond), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids, α-amino acids, and the like.

[0114] Terms such as "amino acid side chain" or "side chain of an amino acid" may be used to refer to a substituent attached to the alpha carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. Amino acid side chains may also include the amino acid side chains described in the context of modified amino acids and / or conjugates described herein.

[0115] The term "conjugated" generally refers to a chemical bond, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In some embodiments, the agent is selected from a half-life extending moiety, a labeling agent, and a therapeutic agent. For half-life extension, for example, the antibody of the present disclosure can be optionally modified to provide an improved pharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, etc.). Modifications that can improve serum half-life are of interest.

[0116] Terms such as "carbohydrate" may be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates such as monosaccharides, disaccharides, etc. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical), or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the subject compounds and conjugates.

[0117] As used herein, the term "isolated" is intended to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. "Isolated" is intended to include a compound within a sample in which the compound of interest has been substantially enriched and / or in which the compound of interest has been partially or substantially purified.

[0118] As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or greater than 98% free from naturally associated components.

[0119] The term "physiological conditions" is meant to encompass conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., that are compatible with living cells.

[0120] "Reactive partner" refers to a molecule or a portion of a molecule that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include the cysteine ​​or serine of a sulfatase motif and formylglycine generating enzyme (FGE), which react to form a reaction product of a converted aldehyde tag that contains formylglycine (fGly) in place of the cysteine ​​or serine in the motif. Other exemplary reactive partners include the aldehyde (e.g., reactive aldehyde group) of the fGly residue of a converted aldehyde tag, and an "aldehyde-reactive reactive partner" that includes an aldehyde-reactive group and a moiety of interest, which react to form a reaction product of a polypeptide having a moiety of interest conjugated to the polypeptide through the fGly residue.

[0121] "N-terminus" refers to the terminal amino acid residue of a polypeptide having a free amine group; the amine group in the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0122] "C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group; the carboxyl group in the non-C-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0123] An "internal site" when used in reference to a polypeptide or the amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or C-terminus.

[0124] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or side effects caused by the disease. "Treatment" as used herein covers any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, e.g., arresting its onset, and (c) relieving the disease, e.g., causing regression of the disease.

[0125] The terms "individual," "subject," "host," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, humans, murines (e.g., rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0126] A "therapeutically effective amount" or "effective amount" refers to the amount of a subject antibody drug conjugate that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the antibody, the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0127] 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.

[0128] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within this 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 stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0130] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of such antibodies, a reference to a "CDR" includes a reference to one or more CDRs and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be written to exclude any optional element. Thus, this statement is intended to serve as a predicate for use of exclusive language such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0131] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication date by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0132] The present disclosure provides antibody conjugates (e.g., antibody drug conjugates (ADCs). The present disclosure also encompasses methods of producing such conjugates, as well as methods of using same. Also provided are compositions comprising the ADCs of the present disclosure, including, in some cases, pharmaceutical compositions. In certain aspects, methods of using an ADC are provided that include administering to an individual a therapeutically effective amount of an ADC of the present disclosure.

[0133] Antibody Drug Conjugates The present disclosure provides conjugates, e.g., antibody drug conjugates (ADCs). By "conjugate" is meant a polypeptide (e.g., an antibody) covalently bonded to a moiety of interest (e.g., a drug or active agent). For example, an antibody drug conjugate according to the present disclosure comprises one or more drugs or active agents covalently bonded to an antibody. In certain embodiments, the polypeptide (e.g., an antibody) and the one or more drugs or active agents are linked to each other through one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds can include a linker as described herein.

[0134] In certain embodiments, the conjugate is a polypeptide conjugate, comprising a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, the one or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest, 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, in which the polypeptide is an antibody, thus providing an antibody drug conjugate. For example, the conjugate can be a drug conjugate, in which the polypeptide is conjugated to one or more drugs or active agents. Various types of drugs and active agents can be used in the conjugates, as described in more detail below.

[0135] One or more drugs or active agents can be conjugated to a polypeptide (e.g., an antibody) at any desired site on the polypeptide. Thus, the present disclosure provides, for example, a polypeptide having a drug or active agent conjugated at a site at or near the C-terminus of the polypeptide. Other examples include polypeptides having a drug or active agent conjugated at a position at or near the N-terminus of the polypeptide. Examples also include polypeptides having a drug or active agent conjugated at a position between the C-terminus and N-terminus of the polypeptide (e.g., at an internal site on the polypeptide). The above combinations are also possible when a polypeptide is conjugated to two or more drugs or active agents.

[0136] 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, the conjugates include polypeptides in which the side chains of one or more amino acid residues in the polypeptide are modified and linked to one or more drugs or active agents (e.g., linked to one or more drugs or active agents through a linker described herein). For example, the conjugates include polypeptides in which the α-carbons of one or more amino acid residues in the polypeptide are modified and linked to one or more drugs or active agents (e.g., linked to one or more drugs or active agents through a linker described herein).

[0137] The embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more moieties, for example, two moieties, three moieties, four moieties, five moieties, six moieties, seven moieties, eight moieties, nine moieties, or ten or more moieties. The moieties may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example, when a polypeptide is conjugated to a first moiety at a first amino acid residue and to two other moieties at a second amino acid residue. Other combinations are possible, such as, but not limited to, a polypeptide conjugated to a first and second moiety at a first amino acid residue and conjugated to a third and fourth moiety at a second amino acid residue.

[0138] One or more amino acid residues of the polypeptide conjugated to one or more moieties of interest can be naturally occurring amino acids, non-natural amino acids, or a combination thereof. For example, the conjugate can include one or more drugs or active agents conjugated to naturally occurring amino acid residues of the polypeptide. In other cases, the conjugate can include one or more drugs or active agents conjugated to non-natural amino acid residues of the polypeptide. One or more drugs or active agents can be conjugated to the polypeptide at a single natural or non-natural amino acid residue as described herein. One or more natural or non-natural amino acid residues in the polypeptide can be conjugated to one or more moieties as described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in the polypeptide can each be conjugated to one or two moieties such that multiple sites in the polypeptide are conjugated to the moiety of interest.

[0139] In certain embodiments, the polypeptide (e.g., an antibody) and the moiety of interest (e.g., a drug or active agent) are conjugated through a conjugation moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bound) to a conjugation moiety, thus indirectly linking the polypeptide and the moiety of interest together through the conjugation moiety. In some cases, the conjugation moiety comprises a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. For example, a general scheme for coupling a moiety of interest to a polypeptide through a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the following general reaction scheme. Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also referred to herein as hydrazino-iso-Pictet-Spengler (HIPS) conjugation moieties and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugation moieties, respectively. [ka]

[0140] In the above reaction scheme, R comprises a moiety of interest (e.g., a drug or active agent) that is conjugated to a polypeptide (e.g., conjugated to a polypeptide through a linker described herein). As shown in the above reaction scheme, a polypeptide that comprises a 2-formylglycine residue (fGly) reacts with a drug or active agent that has been modified to include a conjugation moiety (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to produce a polypeptide conjugate that is attached to the conjugation moiety, thus attaching the drug or active agent to the polypeptide through the conjugation moiety.

[0141] As described herein, the moiety can be any of a variety of moieties, including, but not limited to, a detectable label, or a chemical entity such as a drug or active agent. R' and R" can each independently be any desired substituent, including, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z is CR 21 , N.R. 22 , N, O, or S, where R 21 and R 22 are each independently selected from any of the substituents described for R′ and R″ above.

[0142] As shown in the conjugates and compounds described herein, other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety can be attached (e.g., covalently attached) to a linker. Thus, embodiments of the present disclosure include hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties that are attached to a drug or active agent through a linker. Various embodiments of linkers that can couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker described herein. In other embodiments, the linker is a non-cleavable linker described herein.

[0143] In certain embodiments, a polypeptide (e.g., an antibody) may be conjugated to one or more moieties of interest, and one or more amino acids of the polypeptide are modified prior to conjugation to the moiety of interest. Modification of one or more amino acids of the polypeptide may produce a polypeptide that includes one or more reactive groups suitable for conjugation to the moiety 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 the moiety of interest (e.g., one or more moieties including a conjugation moiety such as a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety described above). For example, an amino acid of the polypeptide may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an "aldehyde tag" or "ald-tag," which as used herein refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR) that has been converted to contain 2-formylglycine residues (referred to herein as "fGly") by the action of formylglycine generating enzyme (FGE). The fGly residue generated 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 (e.g., a sulfatase motif in which a cysteine ​​or serine residue has been converted to fGly by the action of FGE, e.g., L(fGly)TPSR). A converted sulfatase motif can be produced from an amino acid sequence that includes an "unconverted" sulfatase motif (e.g., a sulfatase motif in which the cysteine ​​or serine residue has not been converted to fGly by FGE, but is capable of being converted, e.g., an unconverted sulfatase motif having the sequence L(C / S)TPSR)."Conversion," when used in the context of the action of formylglycine generating enzyme (FGE) on a sulfatase motif, refers to the biochemical modification of a cysteine ​​or serine residue in the sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly or Ser to fGly). Additional aspects of aldehyde tags and their use in site-specific protein modification are described in U.S. Patent Nos. 7,985,783 and 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0144] In some cases, to produce a conjugate, a polypeptide containing an fGly residue can be conjugated to a moiety of interest by reaction of fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above). For example, an fGly-containing polypeptide can be contacted with a reactive partner-containing drug under conditions suitable to provide conjugation of the drug to the polypeptide. In some cases, the reactive partner-containing drug can include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. For example, a drug or active agent can be modified to include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, a drug or active agent is attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, such as covalently attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, through a linker, such as the linkers described in detail herein.

[0145] In certain embodiments, the conjugates of the present disclosure include a polypeptide (e.g., an antibody) having at least one amino acid residue attached to one or more moieties of interest (e.g., a drug or active agent). To generate the conjugate, the amino acid residue of the polypeptide can be modified and then coupled to one or more drugs or active agents attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties described above. In certain embodiments, the amino acid residue of the polypeptide (e.g., an antibody) is a cysteine ​​or serine residue modified to an fGly residue as described above. In certain embodiments, the modified amino acid residue (e.g., an fGly residue) is conjugated to a drug or active agent comprising a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above, providing a conjugate of the present disclosure in which one or more drugs or active agents are conjugated to the polypeptide through the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As used herein, the term fGly' refers to a modified amino acid residue of a polypeptide (eg, an antibody) that is coupled to a moiety of interest (eg, a drug or active agent).

[0146] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one amino acid residue attached to a linker described herein, which in turn is attached to a drug or active agent. For example, the conjugate may comprise a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') conjugated to one or more moieties of interest (e.g., one or more drugs or active agents) described above.

[0147] An embodiment of the present disclosure is a conjugate of formula (I): [ka] During the ceremony, Z is CR 4 or N, R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W 1 But it is a drug, W 2 is an antibody.

[0148] In certain embodiments, Z is CR 4 or N. In certain embodiments, Z is CR 4 In certain embodiments, Z is N.

[0149] 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, and substituted heterocyclyl.

[0150] In certain embodiments, R 1 is hydrogen. In certain embodiments, R 1 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 1 is methyl. In certain embodiments, R 1 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 1 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkynyl or substituted alkynyl, such as a substituted alkenyl. 1 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 1 is C 5~8 Heteroaryl or C 5~8Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 1 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 1 is C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0151] In certain embodiments, R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl.

[0152] In certain embodiments, each R 2is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 2 is hydrogen. In certain embodiments, R 2 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 2 is methyl. In certain embodiments, R 2 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 2 is alkynyl or substituted alkynyl. In certain embodiments, R 2 is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acylamino or aminoacyl. In certain embodiments, R 2 is an alkylamide or a substituted alkylamide. In certain embodiments, R 2is sulfonyl. In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 2 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 2 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 2 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 2 is C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0153] In certain embodiments, each R 3is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 3 is hydrogen. In certain embodiments, R 3 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 3 is methyl. In certain embodiments, R 3 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 3 is alkynyl or substituted alkynyl. In certain embodiments, R 3 is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acylamino or aminoacyl. In certain embodiments, R 3 is an alkylamide or a substituted alkylamide. In certain embodiments, R 3is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 3 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 3 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 3 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 3 is C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0154] In certain embodiments, R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 6-membered heterocyclyl.

[0155] In certain embodiments, each R 4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0156] Each R 4 Various possibilities for R 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 a 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 C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 4 is methyl. In certain embodiments, R 4 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 4 is alkynyl or substituted alkynyl. In certain embodiments, 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 acylamino or aminoacyl. In certain embodiments, R 4 is an alkylamide or a substituted alkylamide. In certain embodiments, R 4 is sulfonyl. In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 4 is C 5~8 Aryl or C 5~8 aryl or substituted aryl (e.g., phenyl or substituted phenyl), such as C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 4 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 4 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 4 is C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0157] In certain embodiments, W 1 is a drug. Further description of drugs can be found in the disclosure herein.

[0158] In certain embodiments, W 2 is an antibody. In certain embodiments, W 2 comprises one or more fGly' residues as described herein. In certain embodiments, the antibody is attached to the remainder of the conjugate through an fGly' residue as described herein. Further description of antibodies used in the subject conjugates is found in the disclosure herein.

[0159] In certain embodiments, the compound of formula (I) comprises a linker L. The linker may be utilized to attach a conjugation moiety (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to one or more moieties of interest. The linker may be attached (e.g., covalently attached) to a conjugation moiety (e.g., as described herein) at any convenient position. For example, the linker may attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety may be used to conjugate the linker (and thus the drug) to a polypeptide, such as an antibody. For example, the conjugation moiety may be used to conjugate the linker (and thus the drug) to a modified amino acid residue of a polypeptide, such as an fGly residue of an antibody.

[0160] In certain embodiments, L is selected from the group consisting of a conjugation moiety, W 1 Thus, the conjugation moiety is linked to W through the linker L. 1 As described above, W 1 is a drug, and thus L attaches the conjugation moiety to the drug, e.g., the conjugation moiety is indirectly attached to the drug through the linker L.

[0161] Any convenient linker may be utilized in the subject conjugates. In certain embodiments, L comprises a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, L comprises an alkyl or substituted alkyl group. In certain embodiments, L comprises an alkenyl or substituted alkenyl group. In certain embodiments, L comprises an alkynyl or substituted alkynyl group. In certain embodiments, L comprises an alkoxy or substituted alkoxy group. In certain embodiments, L comprises an amino or substituted amino group. In certain embodiments, L comprises a carboxyl or carboxyl ester group. In certain embodiments, L comprises an acylamino group. In certain embodiments, L comprises an alkylamido or substituted alkylamido group. In certain embodiments, L comprises an aryl or substituted aryl group. In certain embodiments, L comprises a heteroaryl or substituted heteroaryl group. In certain embodiments, L comprises a cycloalkyl or substituted cycloalkyl group. In certain embodiments, L comprises a heterocyclyl or substituted heterocyclyl group.

[0162] In certain embodiments, L comprises a polymer. For example, the polymer may comprise polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., 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 a polyethylene glycol. Other linkers are possible, as shown in the conjugates and compounds described in more detail below.

[0163] In some embodiments, L is a linker described by the formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 are each independently a linker subunit; a, b, c, d, and f are each independently 0 or 1; and the sum of a, b, c, d, and f is 1 to 6.

[0164] 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.

[0165] In certain embodiments, the linker subunit L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 2 When present, is attached to the drug. In certain embodiments, the linker subunit L 3 When present, is attached to the drug. In certain embodiments, the linker subunit L 4 When present, is attached to the drug. In certain embodiments, the linker subunit L 5 When present, is attached to the drug. In certain embodiments, the linker subunit L 6 If present, is bound to the drug.

[0166] Any convenient linker subunit may be utilized in the linker, L. Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each (if present) independently comprises one or more groups selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0167] In some embodiments, L 1 When present, L 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. 1 comprises polyethylene glycol. In some embodiments, L 1 comprises a modified polyethylene glycol. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L comprises an alkyl group or a substituted alkyl group. 1 comprises an aryl group or a substituted aryl group. In some embodiments, L 1 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0168] In some embodiments, L 2 When present, L 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.2 comprises polyethylene glycol. In some embodiments, L 2 comprises a modified polyethylene glycol. In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L comprises an alkyl group or a substituted alkyl group. 2 comprises an aryl group or a substituted aryl group. In some embodiments, L 2 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0169] In some embodiments, L 3 When present, L 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. 3 comprises polyethylene glycol. In some embodiments, L 3 comprises a modified polyethylene glycol. In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L comprises an alkyl group or a substituted alkyl group. 3 comprises an aryl group or a substituted aryl group. In some embodiments, L 3 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0170] In some embodiments, L 4 When present, L 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. 4 comprises polyethylene glycol. In some embodiments, L 4 comprises a modified polyethylene glycol. In some embodiments, L 4 In some embodiments, L 4In some embodiments, L comprises an alkyl group or a substituted alkyl group. 4 comprises an aryl group or a substituted aryl group. In some embodiments, L 4 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0171] In some embodiments, L 5 When present, L 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. 5 comprises polyethylene glycol. In some embodiments, L 5 comprises a modified polyethylene glycol. In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L comprises an alkyl group or a substituted alkyl group. 5 comprises an aryl group or a substituted aryl group. In some embodiments, L 5 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0172] In some embodiments, L 6 When present, L 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. 6 comprises polyethylene glycol. In some embodiments, L 6 comprises a modified polyethylene glycol. In some embodiments, L 6 In some embodiments, L 6 In some embodiments, L comprises an alkyl group or a substituted alkyl group. 6 comprises an aryl group or a substituted aryl group. In some embodiments, L 6 comprises a diamine (eg, a linking group that comprises an alkylene diamine).

[0173] In some embodiments, L is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - is a linker comprising the formula -(L 1 ) a - is - (T 1 -V 1 ) a - and -(L 2 ) b - is - (T 2 -V 2 ) b - and -(L 3 ) c - is - (T 3 -V 3 ) c - and -(L 4 ) d - is - (T 4 -V 4 ) d - and -(L 5 ) e - is - (T 5 -V 5 ) e - and -(L 6 ) f - is - (T 6 -V 6 ) f - and T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 is a tether group, if present, V 1 , V 2 , V 3 , V 4 , V 5 , and V6 is a covalent bond or linking functional group, if present; a, b, c, d, e, and f each independently represent 0 or 1, and the sum of a, b, c, d, e, and f is 1 to 6.

[0174] As described above, in certain embodiments, L 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 1 is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in formula (I) above). In certain embodiments, V 1 is attached to the drug. In certain embodiments, L 2 When present, T is bound to the drug. 2 If present, it is bound to a drug or V 2 When present, L is attached to the drug. 3 When present, T is bound to the drug. 3 If present, it is bound to a drug or V 3 When present, L is attached to the drug. 4 When present, T is bound to the drug. 4 If present, it is bound to a drug or V 4 When present, L is attached to the drug. 5 When present, T is bound to the drug. 5 If present, it is bound to a drug or V 5 When present, L is attached to the drug. 6 When present, T is bound to the drug. 6 If present, it is bound to a drug or V 6, if present, is bound to the drug.

[0175] Tether group T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 With respect to T, any convenient tether group may be utilized in the subject linkers. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each independently represents a covalent bond, (C 12 ) Alkyl, Substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m Each w is an integer of 1 to 20, each n is an integer of 1 to 30, each p is an integer of 1 to 20, and each m is an integer of 1 to 12.

[0176] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (C1~C 12 ) alkyl or substituted (C1-C 12In certain embodiments, (C1-C 12 )Alkyl is a straight or branched chain alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C1 to C 12 ) Alkyl is C1-C 12 Alkyl or C1-C 10 It can be an alkyl or substituted alkyl, such as an alkyl, C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) alkyl is C2-alkyl. For example, (C1-C 12 ) Alkyl is C1-C 12 Alkylene or C1-C 10 It can be an alkylene or substituted alkylene, such as alkylene, C1-C6 alkylene, or C1-C3 alkylene. In some cases, (C1-C 12 ) Alkyl is C2-alkylene (e.g., CH2CH2).

[0177] In certain embodiments, the substitution (C 12 )Alkyl is a straight or branched chain substituted alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, the substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkyl or substituted C1-C 10 It may be an alkyl, or a substituted alkyl, such as a substituted C1-C6 alkyl, or a substituted C1-C3 alkyl. In some cases, the substituted (C1-C 12 ) alkyl is a substituted C2-alkyl. For example, substituted (C1-C 12 ) Alkyl is a substituted C1-C 12 Alkylene or substituted C1-C 10It can be an alkylene, or a substituted alkylene, such as a substituted C1-C6 alkylene, or a substituted C1-C3 alkylene. In some cases, the substituted (C1-C 12 ) Alkyl is a substituted C2-alkylene.

[0178] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes aryl or substituted aryl. For example, the aryl can be phenyl. In some cases, the substituted aryl is substituted phenyl. Substituted phenyl is (C1-C 12 ) Alkyl, Substituted (C1-C 12 ) may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some cases, the substituted aryl is a substituted phenyl and the substituent comprises a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0179] In some cases, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) includes heteroaryl or substituted heteroaryl. In some cases, the tether group (e.g., T 1 , T 2 , T 3 , T4 , T 5 , and T 6 ) includes cycloalkyl or substituted cycloalkyl. In some cases, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes heterocyclyl or substituted heterocyclyl. In some cases, the substituent on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl includes a cleavable moiety described herein (e.g., an enzymatically cleavable moiety such as a glycoside or glycoside derivative).

[0180] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) comprises an ethylenediamine (EDA) moiety, e.g., an EDA-containing tether group. In certain embodiments, (EDA) w comprises one or more EDA moieties, e.g., w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) moieties may optionally be substituted at one or more convenient positions with any convenient substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety is described by the structure: [ka] In the formula, y is an integer from 1 to 6, r is 0 or 1, and each R 12is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be linked cyclically to form, for example, a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 In certain embodiments, y is 1 and the adjacent R 12 The groups are selected from hydrogen, alkyl (eg, methyl), and substituted alkyl (eg, lower alkyl-OH such as ethyl-OH or propyl-OH).

[0181] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be optionally substituted at one or more convenient positions with any convenient substituent, for example, alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure: [ka] In the formula, R 12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxy-modified polyethylene glycol.

[0182] In certain embodiments, R 12 is represented by the formula: (PEG) k which may be represented by the following structure: [ka] wherein k is an integer from 1 to 20, e.g., 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, k is 2. In certain embodiments, R 17 is selected from OH, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 17 is COOH. In certain embodiments, R 17 is COOCH3.

[0183] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (PEG) n (PEG) n is a polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG) n is described by the following structure: [ka] In the formula, n is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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.

[0184] In certain embodiments, a tether group (e.g., T 1 , T2 , T 3 , T 4 , T 5 , and / or T 6 ) is (AA) p where AA is an amino acid residue. Any convenient amino acid may be utilized. Amino acids of interest include, but are not limited to, L-amino acids and D-amino acids, naturally occurring amino acids such as any of the 20 major 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, and the like. In certain embodiments, p is an integer between 1 and 50, e.g., between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 12, or between 1 and 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0185] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6) includes amino acid analogs. Amino acid analogs include compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D forms, as well as L forms of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, with the difference(s) being one or more modified groups in the molecule. Such modifications may include, but are not limited to, replacing an atom (such as N) with a related atom (such as S), adding a group (such as methyl or hydroxyl) or an atom (such as Cl or Br), deleting a group, replacing a covalent bond (replacing a single bond with a double bond), or a combination thereof. For example, amino acid analogs may include alpha-hydroxy acids, alpha-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.

[0186] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is the formula -(CR 13 OH) m -, where m is 0 or n is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, each R 13is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 13 is hydrogen. In certain embodiments, R 13 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 13 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. In certain embodiments, R 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 13 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 13 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 13 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 13 is C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0187] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are selected from R 13 As described above.

[0188] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T4 , T 5 , and / or T 6 ) includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).

[0189] In some embodiments, the tether group comprises a MABO group, described by the following structure: [ka]

[0190] In some embodiments, the tether comprises an MABC group, described by the following structure: [ka]

[0191] In some embodiments, the tether comprises a PABO group described by the following structure: [ka]

[0192] In some embodiments, the tether comprises a PABC group, described by the following structure: [ka]

[0193] In some embodiments, the tether comprises a PAB group described by the following structure: [ka]

[0194] In some embodiments, the tether comprises a PABA group described by the following structure: [ka]

[0195] In some embodiments, the tether comprises a PAP group described by the following structure: [ka]

[0196] In some embodiments, the tether comprises a PHP group described by the following structure: [ka]

[0197] In certain embodiments, each R 14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0198] In certain embodiments, R 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 14is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C 2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 14 is alkynyl or substituted alkynyl. In certain embodiments, R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14 is acyl or acyloxy. In certain embodiments, R 14 is acylamino or aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 14 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 14 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 14 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 14 For example, C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C 3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0199] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring can be substituted with one or more additional groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0200] In certain embodiments of the linker L, the tether group T 1 , T 2 , T 3 , T 4 , T 5 , or T 6 is optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0201] 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 glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0202] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] may be selected from:

[0203] Linking functional group V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 With respect to, any convenient linking functional group may be utilized in the linker L. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphorophosphoramidate, thiophosphoraidate, and the like. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, -P(O)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0204] In some embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0205] In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is C 1~6 Alkyl or C 1~6 Substituted alkyl, or C 1~4 Alkyl or C 1~4 Substituted alkyl, or C 1~3 Alkyl or C 1~3 In certain embodiments, R is an alkyl or substituted alkyl, such as a substituted alkyl. 15 is C 2~6 Alkenyl or C 2~6 Substituted alkenyl, or C 2~4 Alkenyl or C 2~4 Substituted alkenyl, or C 2~3 Alkenyl or C2~3 In certain embodiments, R is an alkenyl or substituted alkenyl, such as a substituted alkenyl. 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 15 is C 5~8 Aryl or C 5~8 aryl or substituted aryl, such as C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl. In certain embodiments, R 15 is C 5~8 Heteroaryl or C 5~8 Heteroaryl or substituted heteroaryl, such as C5 heteroaryl or C5 substituted heteroaryl, or C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R 15 is C 3~8 Cycloalkyl or C 3~8 Substituted cycloalkyl, e.g., C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as substituted cycloalkyl. 15 is C 3~8 Heterocyclyl or C 3~8 Substituted heterocyclyl, for example, C3~6 Heterocyclyl or C 3~6 substituted heterocyclyl, or C 3~5 Heterocyclyl or C 3~5 Heterocyclyl or substituted heterocyclyl, such as substituted heterocyclyl.

[0206] In certain embodiments, each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl can be independently selected from R 15 is as described above.

[0207] In certain embodiments, the tether group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises a hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

[0208] As described above, in some embodiments, L is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T6 -V 6 ) f -, wherein a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is 1 to 6.

[0209] In some embodiments, in the linker L, T 1 is (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 are each independently: (C1 to C 12 ) Alkyl, Substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15SO2-, and -P(O)OH-, where q is an integer from 1 to 6; (PEG) n teeth, [ka] wherein n is an integer from 1 to 30; EDA has the following structure: [ka] where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is [ka] and AA is an amino acid residue, and p is an integer from 1 to 20. Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring, Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0210] In certain embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V3 , V 4 , V 5 , and V 6 is selected from: T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 is -CO-, T 3 is (C1~C 12 ) alkyl, V 3 is -CO-, and d, e, and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 does not exist, T 3 is (PEG) n and V 3 is -CO-, and d, e, and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 does not exist, T 3 is (PEG) n and V 3 is -CO-, T 4 is (AA) p and V 4 does not exist, T 5 is PABC, and V 5 does not exist, and f is 0, or T 1 is (C1~C 12 ) alkyl, V1 is -CONH-, T 2 is (PEG) n and V 2 is -CO-, T 3 is (AA) p and V 3 does not exist, T 4 is PABC, and V 4 does not exist, and e and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is an amino acid analogue, V 2 is -NH-, T 3 is (PEG) n and V 3 is -CO-, T 4 is (AA) p and V 4 does not exist, T 5 is PABC, and V 5 does not exist, and f is 0.

[0211] For example, in certain embodiments, the linker L of formula (I) is: [ka] TIFF2024529466000021.tif151149.

[0212] In certain embodiments, the wavy line [ka] The left side of the linker L, denoted by 1 For example, the right side of the linker L is linked to the drug W through an amide bond. 1 can bind to

[0213] In certain embodiments, the conjugate of formula (I) is: [ka] The structure is selected from:

[0214] In certain embodiments, the conjugate is an antibody-drug conjugate, as described above, in which the antibody and the drug are linked together by a linker (e.g., L). In some cases, the linker is a non-cleavable linker.

[0215] In other cases, the linker is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, which include one or more bonds that can dissociate under certain conditions and thus separate the cleavable linker into two or more separable parts. For example, the cleavable moiety can include one or more covalent bonds that can dissociate or decompose under certain conditions and separate the cleavable linker into two or more parts. Thus, a cleavable linker can be included in an antibody-drug conjugate such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at the desired target site of action of the drug.

[0216] In some cases, the cleavable linker comprises two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties may be configured such that cleavage of both cleavable moieties is required to separate or release the drug from the antibody at the desired target site of action of the drug. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that prevents cleavage of the first cleavable moiety. By "prevents cleavage" it is meant that the presence of the uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage, thus substantially reducing the amount of cleavage of the cleavable linker or preventing cleavage. For example, the presence of the uncleaved second cleavable moiety can prevent cleavage of the first cleavable moiety. The prevention of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount of or prevents release of the drug from the antibody, for example, can substantially reduce or prevent premature release of the drug from the antibody until the antibody drug conjugate is at or near the desired target site of action of the drug.

[0217] In some cases, since the second cleavable moiety interferes with the cleavage of the first cleavable moiety, cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the interference with the cleavage of the first cleavable moiety, thus allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety can dissociate or separate the cleavable linker into two or more moieties as described above, releasing the drug from the antibody drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially, it is meant that in the presence of an uncleaved second cleavable portion, about 10% or less cleavage of the first cleavable portion occurs, for example, in the presence of an uncleaved second cleavable portion, about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable portion occurs.

[0218] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the drug's desired target site of action. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thus allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, and thus separating or releasing the drug from the antibody at the drug's desired target site of action, as described above. In certain cases, cleavage of the second cleavable moiety exposes the first cleavable moiety for subsequent cleavage, but cleavage of the second cleavable moiety does not by itself result in cleavage of the cleavable linker (e.g., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0219] Each of the cleavable moieties included in the cleavable linker can be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety, and the second cleavable moiety can be a second enzymatically cleavable moiety. The enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above through the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved through the enzymatic action of an enzyme, such as, but not limited to, a peptide, a glycoside, etc. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired target site of action, such as the desired target site of action of the drug released from the antibody drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in significant amounts in other regions, such as whole blood, plasma, or serum. Thus, the cleavage of the enzymatically cleavable moiety can be controlled so that substantial cleavage occurs at the desired site of action, while cleavage does not occur significantly in other regions or before the antibody drug conjugate reaches the desired site of action.

[0220] For example, as described herein, the antibody drug conjugates of the present disclosure can be used for the treatment of cancer, such as for the delivery of a cancer therapeutic to a desired site of action where cancer cells are present. In some cases, an enzyme, such as the protease enzyme cathepsin B, can be a biomarker for cancer, which is overexpressed in cancer cells. The overexpression, and therefore localization, of a particular enzyme in cancer can be used in conjunction with an enzymatically cleavable moiety included in the cleavable linker of the antibody drug conjugates of the present disclosure to specifically release the drug at a desired site of action (e.g., the site of the cancer (and the overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme that is overexpressed in cancer cells. For example, the enzyme can be the protease enzyme cathepsin B. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by a protease enzyme, such as cathepsin B.

[0221] In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide suitable for use in a cleavable linker and capable of being cleaved through the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzymatically cleavable moieties include, for example, Val-Ala, Phe-Lys, and the like. For example, the first cleavable moiety described above (i.e., the cleavage 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 (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), thus substantially reducing or preventing premature release of the drug from the antibody until the antibody drug conjugate is at or near the desired target site of action of the drug. In some cases, one of the amino acid residues of the peptide that comprises the first cleavable moiety is linked to or comprises a substituent, and the substituent comprises a second cleavable moiety. In some cases, the second cleavable moiety comprises a glycoside.

[0222] In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycosyl). In some cases, the glycoside can promote an increase in hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside. The glycoside can be any glycoside or glycoside derivative that is suitable for use in a cleavable linker and can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside. For example, in some embodiments, the first cleavable moiety comprises a peptide and the second cleavable moiety comprises a glycoside. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. In some 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.

[0223] The glycoside can be attached (e.g., covalently attached) to the cleavable linker through a glycosidic bond. The glycosidic bond can be linked to the cleavable linker through various types of bonds, such as, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside can be cleaved from the cleavable linker to which it is attached by an enzyme (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). The glycoside can be removed or cleaved from the cleavable linker by any convenient enzyme that can perform cleavage (hydrolysis) of the glycosidic bond that connects the glycoside to the cleavable linker. An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that connects the glycoside to the cleavable linker is a glucuronidase, glycosidase, such as galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes can also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that connects the glycoside to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that connects the glycoside 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 can be a lysosomal enzyme, such as a lysosomal glycosidase, that is 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 that is found at or near the target site where the enzyme that mediates the cleavage of the first cleavable moiety is found.

[0224] Any of the chemical entities, drugs, linkers, and coupling moieties depicted in the descriptions and structures set forth herein can be adapted for use in the subject conjugates.

[0225] Additional disclosure regarding hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compounds and methods for producing the conjugates can be found in U.S. Patent Nos. 9,310,374 and 9,493,413, the disclosures of each of which are incorporated herein by reference. Additional disclosure regarding cleavable linkers can be found in PCT Publication No. WO2020 / 154437, filed January 22, 2020, and U.S. Application No. 17 / 531,343, filed November 19, 2021, the disclosures of each of which are incorporated herein by reference.

[0226] Antibody drug conjugates (ADCs) generally comprise an antibody linked to a drug, such as a cytotoxic small molecule, and are targeted in non-healthy cells. The target antigen may be expressed in both non-healthy and healthy cells in vivo, so the payload (e.g., a drug or active agent) can be offloaded on either type of cell. In this case, the ADC may target off-target cells or healthy cells that express the same antigen as the non-healthy cells. This may result in what is called cross-reactivity, which can be detected clinically. For example, administration of an ADC to a subject may induce toxicity associated with the target-mediated cross-reactivity of the ADC. Toxicity may imply a limited dosage that can be administered to a subject, regardless of the specificity or efficacy of the ADC itself. Thus, in some cases, it may be desirable to reduce the toxicity caused by the cross-reactivity of the ADC with healthy cells that express the target antigen.

[0227] In some cases, the ADC of the present disclosure reduces toxicity associated with target-mediated cross-reactivity of the ADC when the ADC is administered to a subject. For example, the ADC of formula (I) described herein may reduce or reduce toxicity caused by target-mediated cross-reactivity in a subject compared to toxicity caused by cross-reactivity when the subject is administered an ADC that is not formula (I). Reducing or reducing toxicity refers to reducing or reducing one or more of the parameters associated with toxicity in a subject. For example, the parameters can be scored based on clinical observations and can correspond to a body area or a functional, physiological, or behavioral aspect in a subject. By reducing toxicity, the ADC of the present disclosure reduces or reduces the occurrence, intensity, severity, and / or duration of a reaction parameter in a subject based on the clinical score(s) of each body area or physiological or behavioral aspect of the subject. Parameters related to toxicity in a subject may include, but are not limited to, activity level / uninduced behavior, induced behavior, motor / neurological, respiration, posture, physical condition, skin condition, eye condition, tumors or infections (irrelevant to disease indication), body weight, and the like, and combinations thereof.

[0228] In some cases, ADC other than formula (I) as used herein refers to ADC in which the linker payload is different from the ADC of formula (I) disclosed herein, either structurally or functionally, or both. In some cases, ADC other than formula (I) is not encompassed by formula (I) of the present disclosure. For example, ADC other than formula (I) may refer to an antibody linked to a drug with a linker having a different structure compared to formula (I).

[0229] In some embodiments, when an ADC of Formula (I) is administered to a subject, target-mediated cross-reactivity is reduced in the subject by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more. In some embodiments, when an ADC of Formula (I) is administered to a subject, target-mediated cross-reactivity is decreased in the subject by reducing the number, severity, and / or duration of clinical observations of a particular parameter or combination of parameters described above.

[0230] In some embodiments, when an ADC of Formula (I) is administered to a subject, the stability of the ADC in vivo is increased compared to when the subject is administered an ADC other than of Formula (I) and the target antigen is the same.

[0231] In some embodiments, as discussed above, an ADC of formula (I) comprises a cleavable linker having a first and a second cleavable moiety, and the presence of the uncleaved second cleavable moiety protects the first cleavable moiety from cleavage, thus substantially reducing or preventing release of the drug from the ADC. For example, in some embodiments, an ADC of formula (I) comprises a cleavable linker, and the second cleavable moiety (e.g., a cleavable moiety that protects the first cleavable moiety from premature cleavage) is a glycoside or glycoside derivative, and the first cleavable moiety comprises a peptide. In some embodiments, the subject may have differential expression of glucuronidase or glycosidase in healthy cells compared to non-healthy cells targeted by the ADC. For example, healthy cells may express less glucuronidase or glycosidase compared to non-healthy cells targeted by the ADC. In some cases, where there is target-mediated cross-reactivity of the ADC with healthy cells expressing the target antigen, reducing expression of glucuronidase or glycosidase by the healthy cells may result in reducing or preventing cleavage of the second cleavable portion of the linker and thus reducing or preventing release of the drug from the ADC at the site of the healthy cells. This in turn, in some embodiments, may result in reduced toxicity caused by target-mediated cross-reactivity of the ADC with healthy cells expressing the target antigen.

[0232] Compounds useful for producing conjugates The present disclosure provides hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compounds useful for producing the conjugates described herein. In certain embodiments, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compounds can be useful conjugation moieties for the conjugation of a polypeptide (e.g., an antibody) and a drug or active agent (e.g., a camptothecin or camptothecin derivative). For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound can be bound to a polypeptide (antibody) and also to a drug or active agent, thus indirectly binding both the polypeptide (antibody) and the drug.

[0233] In certain embodiments, the compound is a compound of formula (III): [ka] During the ceremony, Z is CR 4 or N, R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W 1 But it is a drug, a compound.

[0234] With respect to the compounds of formula (III), the substituents Z, R 2 , R 3 , R 4 , L, and W 1 is as described above for the conjugate of formula (I). Similarly, for the linker L of formula (III), T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 The substituents are as described above for the conjugates of formula (I).

[0235] For example, in some cases of compounds of formula (III), T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is selected from: T 1 is (C1~C12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 is -CO-, T 3 is (C1~C 12 ) alkyl, V 3 is -CO-, and d, e, and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 does not exist, T 3 is (PEG) n and V 3 is -CO-, and d, e, and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is 4AP, V 2 does not exist, T 3 is (PEG) n and V 3 is -CO-, T 4 is AA and V 4 does not exist, T 5 is PABC, and V 5 does not exist, and f is 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CONH-, T 2 is (PEG) n and V 2 is -CO-, T 3is AA and V 3 does not exist, T 4 is PABC, and V 4 does not exist, and e and f are each 0, or T 1 is (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is an amino acid analogue, V 2 is -NH-, T 3 is (PEG) n and V 3 is -CO-, T 4 is AA and V 4 does not exist, T 5 is PABC, and V 5 does not exist, and f is 0.

[0236] For example, in certain embodiments, the linker L of formula (III) is: [ka] The structure is selected from:

[0237] In certain embodiments, the wavy line [ka] The left side of the linker L, denoted by 1 For example, the right side of the linker L is linked to the drug W through an amide bond. 1 can bind to

[0238] Compounds of formula (III) can be used in the conjugation reactions described herein, in which a drug or active agent attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is conjugated to a polypeptide (e.g., an antibody) to form an antibody drug conjugate.

[0239] In certain embodiments, the compound of formula (III) is: [ka] It has a structure selected from TIFF2024529466000028.tif74160.

[0240] Any of the chemical entities, linkers and conjugating moieties described in the structures above can be adapted for use in the subject compounds and conjugates.

[0241] antibody As noted above, the subject conjugates include a substituent W 2 As used herein, amino acids may be referred to by their standard name, their standard three letter abbreviation, and / or their standard one letter abbreviation, such as alanine or Ala or A, cysteine ​​or Cys or C, aspartic acid or Asp or D, glutamic acid or Glu or E, phenylalanine or Phe or F, glycine or Gly or G, histidine or His or H, isoleucine or Ile or I, lysine or Lys or K, leucine or Leu or L, methionine or Met or M, asparagine or Asn or N, proline or Pro or P, glutamine or Gln or Q, arginine or Arg or R, serine or Ser or S, threonine or Thr or T, valine or Val or V, tryptophan or Trp or W, and tyrosine or Tyr or Y.

[0242] The antibodies of the present disclosure may bind to specific target cells (e.g., cancerous tissues) and may not exhibit binding to normal tissues (e.g., insignificant binding as measured by immunohistochemistry or undetectable binding by immunohistochemistry). For example, the antibodies described herein may bind to human stomach, breast, and / or lung tissues that have cancerous cells, while not exhibiting detectable binding to human stomach, breast, and / or lung tissues that do not have cancerous cells.

[0243] The antibodies are used in a variety of research, diagnostic, and therapeutic applications, including for performing any of the methods described in U.S. Application Publication Nos. 2012 / 0141375 and 2016 / 0145343, the disclosures of each of which are incorporated herein by reference.

[0244] The subject antibodies exhibit high affinity binding to their specific targets. For example, the subject antibodies have an affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M or 10 -12 For example, the subject antibodies can bind with an affinity of about 10 -7 M ~ about 10 -8 M, about 10 -8 M ~ about 10 -9 M, about 10 -9 M ~ about 10 -10 M, about 10 -10 M ~ about 10 -11 M, or about 10 -11 M ~ about 10 -12 M or 10 -12 It can bind to the epitope with an affinity of greater than M.

[0245] As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as a number of immunoglobulin variable region genes. A full-length immunoglobulin light chain (about 25 kD 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. A full-length immunoglobulin heavy chain (about 50 kD or 446 amino acids) is encoded by a variable region gene (about 116 amino acids) at the N-terminus and one of the other aforementioned constant region genes at the C-terminus, e.g., gamma (encoding about 330 amino acids). In some embodiments, the subject antibodies comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0246] In some embodiments, the subject antibodies do not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but instead comprise an antigen-binding fragment of a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain. In some embodiments, the antigen-binding fragments are comprised on separate polypeptide chains, while in other embodiments, the antigen-binding fragments are comprised within a single polypeptide chain. The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that are capable of specifically binding to a target, as described above. Examples of binding fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment (consisting of the VH and CH1 domains), (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody), (v) a dAb fragment (consisting of the VH), (vi) an isolated CDR, and (vii) a single chain Fv (scFv) (where the VH and VL domains are linked by a synthetic linker using recombinant means so as to pair to form a monovalent molecule). (viii) diabodies (composed of two scFvs linked such that the VH and VL domains do not pair to form a monovalent molecule, and the VH of each one of the scFvs pairs with the VL domain of the other scFv to form a divalent molecule); and (ix) diabodies (composed of at least two antigen-binding regions, each region binding to a different epitope). In some embodiments, a subject antibody fragment is a Fab fragment. In some embodiments, a subject antibody fragment is a single chain antibody (scFv).

[0247] In some embodiments, the subject antibodies are recombinant or modified antibodies, such as chimeric, humanized, deimmunized, or in vitro generated antibodies. The term "recombinant" or "modified" antibodies as used herein is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell, (ii) antibodies isolated from a recombinant combinatorial human antibody library, (iii) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or (iv) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized antibodies, CDR-grafted antibodies, chimeric antibodies, deimmunized antibodies, and in vitro generated antibodies, and can optionally include constant regions derived from human germline immunoglobulin sequences.

[0248] Full-length bispecific antibodies can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions in the heavy chain CH3 interface of each half molecule to favor heterodimerization of two antibody half molecules with distinctly different specificities, either in vitro in a cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation / association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation / association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding to a distinct epitope.

[0249] The "knobs-in-holes" strategy (see, for example, PCT International Publication No. WO2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knobs and holes are (represented 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, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S / L368A / Y407V.

[0250] Other strategies may be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in US Patent Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, and 2011 / 0123532. In another strategy, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y / F405A / Y407V / T394W, T366I / K3, as described in US Patent Publication Nos. 2012 / 0149876 or 2013 / 0195849. 92M / T394W / F405A / Y407V, T366L / K392M / T394W / F405A / Y407V, L351Y / Y407A / T366A / K409F, L351Y / Y407A / T366V / K409F, Y407A / T366A / K409F, or T350V / L351Y / F405A / Y407V, T350V / T366L / K392L / T394W.

[0251] Also provided are single chain bispecific antibodies. In some embodiments, the single chain bispecific antibodies of the present disclosure are bispecific scFvs. The subject antibodies can be humanized. The constant region(s), if present, can also be substantially or completely derived from a human immunoglobulin.

[0252] Methods for making humanized antibodies are known in the art. The replacement of mouse CDRs into a human variable domain framework can result in the retention of their correct spatial orientation, for example, in the human variable domain framework adopting the same or similar conformation as the mouse variable framework from which the CDRs originated. This can be achieved by obtaining the human variable domain from a human antibody whose framework sequences show a high degree of sequence identity with the mouse variable framework domain from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies.

[0253] Having identified the complementarity determining regions of the mouse donor immunoglobulin and the appropriate human acceptor immunoglobulin, the next step is to determine which, if any, residues from these components should be replaced to optimize the properties of the resulting humanized antibody. In general, substitution of human amino acid residues with mouse should be minimized, since the introduction of mouse residues increases the risk of the antibody eliciting a human anti-mouse antibody (HAMA) response in humans. To monitor HAMA responses in a particular patient or during clinical trials, art-recognized methods of determining immune responses can be performed. Patients administered humanized antibodies can undergo immunogenicity assessments at the start of and throughout administration of the therapy. HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients, using methods known in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0254] Specific amino acids from the human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and / or binding to antigen. The non-natural juxtaposition of the mouse CDR regions with the human variable framework regions may result in non-natural conformational constraints that result in loss of binding affinity unless corrected by substitution of specific amino acid residues. The selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for creating three-dimensional images of immunoglobulin molecules are known in the art. In general, molecular models are created starting from the resolved structure of an immunoglobulin chain or domain thereof. The chains to be modeled are compared for amino acid sequence similarity with the chains or domains of the resolved three-dimensional structure, and the chains or domains showing the greatest sequence similarity are selected as the starting point for construction of the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, preferably those sharing at least 60%, 70%, 80%, 90% sequence identity are selected for modeling. The solved starting structure is modified to take into account differences between the actual amino acids in the immunoglobulin chain or domain being modeled and the amino acids in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0255] When the framework residues defined by Kabat constitute the structural loop residues defined by Chothia, amino acids present in the mouse antibody can be selected for substitution into the humanized antibody. Residues "adjacent to the CDR region" include amino acid residues in positions immediately adjacent to one or more of the CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., CDRs defined by Kabat or CDRs defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs, which, if selected from the acceptor, would distort the donor CDR and reduce affinity. Furthermore, adjacent amino acids may directly interact with the antigen (see, e.g., Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to preserve all antigen contacts that provide affinity in the original antibody.

[0256] In some embodiments, a subject antibody comprises an scFv multimer. For example, in some embodiments, a subject antibody is an scFv dimer (e.g., comprising two tandem scFvs (scFv2)), an scFv trimer (e.g., comprising three tandem scFvs (scFv3)), an scFv tetramer (e.g., comprising four tandem scFvs (scFv4)), or a multimer of more than four scFvs (e.g., in tandem). The scFv monomers can be linked in tandem via a linker that is 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 , glycine-serine polymers, and the like.

[0257] In some embodiments, the subject antibodies comprise an immunoglobulin constant region (e.g., Fc region). The Fc region, if present, can be a human Fc region. If a constant region is present, the antibody can comprise both a light chain constant region and a heavy chain constant region. The antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is human isotype IgG1Fc. The light chain constant region can be lambda or kappa. The subject antibodies (e.g., the subject humanized antibodies) can comprise sequences from more than one class or isotype. The antibodies can be expressed as tetramers comprising two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab'F(ab')2, and Fv, or as single chain antibodies in which the heavy and light chain variable domains are linked through a spacer.

[0258] In some embodiments, an antibody of the disclosure may include one or more amino acid substitutions introduced into the Fc region. In some embodiments, one or more of the amino acid substitutions may be at positions 239, 298, 326, 330, and 332 in the Fc region. In some embodiments, an 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.

[0259] In some embodiments, the subject antibodies comprise one or more non-naturally occurring amino acids. In some embodiments, the non-naturally occurring amino acids comprise carbonyl, acetyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, or alkyne groups. The inclusion of non-naturally occurring amino acids can provide linkage to polymers, second polypeptides, scaffolds, and the like. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0260] The present disclosure also provides antibodies with a binding moiety of interest, e.g., a detectable label, a drug, a half-life extending moiety, etc. Modification of the antibody can be accomplished by a variety of synthetic and / or recombinant methods. The moiety or moieties attached to the antibody can provide one or more of a wide variety of functions or characteristics. Exemplary moieties include detectable labels (e.g., dye labels (e.g., chromophores, fluorophores), biophysical probes (spin labels, nuclear magnetic resonance (NMR) probes), fluorescence resonance energy transfer (FRET) type labels (e.g., at least one member of a FRET pair, 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 tags (e.g., FLAG, His(6), etc.), water soluble polymers (e.g., PEGylation), purification tags (e.g., to facilitate isolation by affinity chromatography (e.g., binding of a FLAG epitope, membrane localization domains (e.g., lipid or glycophosphatidylinositol (GPI)-type anchors), immunization tags (e.g., to facilitate binding of a polypeptide to a surface, including selective binding), drugs (e.g., to facilitate drug targeting, e.g., through binding of a drug to an antibody), and the like.

[0261] The subject antibodies can be glycosylated, e.g., the subject antibodies can include covalently bound carbohydrate or polysaccharide moieties. Glycosylation of antibodies is typically either N-linked or O-linked. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence such that it contains an N- or O-linked glycosylation site. Similarly, removal of glycosylation sites can be accomplished by altering amino acids within the native glycosylation sites of the antibody.

[0262] The subject antibodies, in some embodiments, include a "radiopaque" label, e.g., a label that can be easily visualized, e.g., using an X-ray. Radiopaque materials are known to those of skill in the art. The most common radiopaque materials include iodide, bromide, or barium salts. Other radiopaque materials are also known, including, but not limited to, organobismuth derivatives, radiopaque multi-urethanes, organobismuth complex materials, radiopaque barium multimer complexes, and the like.

[0263] Methods for the modification of antibodies The antibody conjugates of the present disclosure comprise 1) an Ig heavy chain constant region conjugated to a moiety of interest and an Ig light chain constant region conjugated to a moiety of interest, 2) an Ig heavy chain constant region conjugated to a moiety of interest and an Ig light chain constant region not conjugated to a moiety of interest, or 3) an Ig heavy chain constant region not conjugated to a moiety of interest and an Ig light chain constant region conjugated to a moiety of interest. The subject antibody conjugates can also comprise a VH and / or VL domain conjugated to a moiety of interest.

[0264] In one example, an antibody can be modified to contain a 2-formylglycine residue, which can serve as a chemical handle for attachment of a heterologous moiety. For example, the heavy and / or light chain constant regions of an antibody of the present disclosure can be modified to contain an amino acid sequence of a sulfatase motif that can be converted to contain 2-formylglycine (fGly) by the action of 2-formylglycine generating enzyme (FGE). Such a sulfatase motif can also be referred to herein as an FGE modification site. The action of FGE is directed in a sequence-specific manner in that FGE acts at a sulfatase motif located within an immunoglobulin polypeptide. The moiety of interest is provided as a component of a reactive partner for reaction with the aldehyde of the fGly residue of the converted aldehyde tag of the tagged Ig polypeptide. A wide range of commercially available reagents can be used to achieve attachment of the moiety of interest to the fGly residue of the aldehyde-tagged Ig polypeptide. For example, aminooxy, hydrazide, or thiosemicarbazide derivatives of some of the moieties of interest are suitable reactive partners and are readily available or can be generated using standard chemical methods.

[0265] As noted above, the amino acid sequence of the antibody can be modified to contain a sulfatase motif that includes a serine or cysteine ​​residue that can be converted (oxidized) to a 2-formylglycine (fGly) residue by the action of formylglycine generating enzyme (FGE), either in vivo (e.g., during translation of an aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting an aldehyde-tagged protein with FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

[0266] Sulfatase motif The minimal sulfatase motif of the aldehyde tag is usually 5 or 6 amino acid residues in length, and usually not more than 6 amino acid residues in length. The sulfatase motifs provided in the Ig polypeptides are at least 5 or 6 amino acid residues in length, and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, defining sulfatase motifs that are less than 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acid residues in length.

[0267] In certain embodiments, the polypeptides of interest include those in which one or more amino acid residues have been inserted, deleted or substituted (replaced), for example, 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues, compared to the native amino acid sequence to provide the sequence of the sulfatase motif in the polypeptide. In certain embodiments, the polypeptides include modifications (insertions, additions, deletions and / or substitutions / replacements) 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 compared to the native amino acid sequence of the polypeptide. Where the native amino acid sequence of a polypeptide (e.g., an antibody) contains one or more residues of a desired sulfatase motif, the total number of residue modifications may be reduced, for example, by site-specific modification (insertion, addition, deletion, substitution / replacement) of amino acid residues adjacent to the native amino acid residues to provide the sequence of the desired sulfatase motif. In certain embodiments, the extent of modification of the native amino acid sequence of the target polypeptide is minimized, so as to minimize the number of amino acid residues inserted, deleted, substituted (replaced), or added (e.g., to the N-terminus or C-terminus). Minimizing the extent of amino acid sequence modification of the target polypeptide may minimize the effects that such modifications may have on function and / or structure.

[0268] It should be noted that while aldehyde tags of particular interest are those that include at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it is readily understood that longer aldehyde tags are contemplated and encompassed by the present disclosure and may be used in the compositions and methods of the present disclosure. Thus, the aldehyde tag may include a minimal sulfatase motif of 5 or 6 residues, or may be longer, including a minimal sulfatase motif where additional amino acid residues may flank the motif on the N-terminus and / or C-terminus. For example, aldehyde tags of 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.

[0269] The aldehyde tag may be present at or near the C-terminus of an Ig heavy chain, for example, the aldehyde tag may be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a native wild-type Ig heavy chain. The aldehyde tag may be present in the CH1 domain of an Ig heavy chain. The aldehyde tag may be present in the CH2 domain of an Ig heavy chain. The aldehyde tag may be present in the CH3 domain of an Ig heavy chain. The aldehyde tag may 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.

[0270] In certain embodiments, the sulfatase motif used can be described by the following formula: X 1 Z 1 X 2 Z 2 X 3 Z 3 (SEQ ID NO: 13) (I'), wherein Z 1 is cysteine ​​or serine (which may also be represented by (C / S)), Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3is a basic amino acid (e.g., arginine (R), which can be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), usually A, G, L, V, or I; X 1 is present or absent, and if present, can be any amino acid, but typically is an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), typically L, M, V, S, or T, more typically L, M, S, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can independently be any amino acid, but typically can be an aliphatic amino acid, a polar uncharged amino acid, or a sulfur-containing amino acid (e.g., other than an aromatic or charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G. In one example, the aldehyde tag is of the formula L(C / S)TPSR (SEQ ID NO: 14), e.g., LCTPSR (SEQ ID NO: 15) or LSTPSR (SEQ ID NO: 16). Thus, the present disclosure provides antibodies comprising an aldehyde-tagged Ig heavy chain and / or an aldehyde-tagged Ig light chain, wherein the aldehyde-tagged Ig antibody comprises an Ig constant region, wherein the amino acid sequence of the heavy and / or light chain comprises such a sulfatase motif.

[0271] For example, in some embodiments, the amino acid sequence of the antibody heavy and / or light chain is represented by the formula X 1 Z 1 X 2 Z 2 X 3 Z 3 and can be modified to provide a sequence of at least five amino acids of Z 1 is cysteine ​​or serine, Z 2 is a proline or alanine residue, Z3 is an aliphatic amino acid or a basic amino acid, X 1 is present or absent, and if present, is any amino acid, except that when the heterologous sulfatase motif is at the N-terminus of the polypeptide, X 1 Provided that there exists X 2 and X 3 are each independently any amino acid; This sequence is within or adjacent to a solvent accessible loop region of the Ig constant region, and this sequence is not at the C-terminus of the Ig heavy chain.

[0272] The sulfatase motif is generally selected such that it is capable of being converted by a selected FGE, e.g., an FGE present in the host cell in which the aldehyde-tagged polypeptide is expressed, or an FGE contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0273] For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including human FGE), the sulfatase motif can be of the following formula: X 1 CX 2 PX 3 Z 3 (I'') During the ceremony, X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G; Z 3 is a basic amino acid (e.g., arginine (R), which can 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.

[0274] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO:17), MCTPSR (SEQ ID NO:18), VCTPSR (SEQ ID NO:19), LCSPSR (SEQ ID NO:20), LCAPSR (SEQ ID NO:21), LCVPSR (SEQ ID NO:22), LCGPSR (SEQ ID NO:23), ICTPAR (SEQ ID NO:24), LCTPSK (SEQ ID NO:25), MCTPSK (SEQ ID NO:26), VCTPSK (SEQ ID NO:27), LCSPSK (SEQ ID NO:28), LCAPSK (SEQ ID NO:29), LCVPSK (SEQ ID NO:30), LCGPSK (SEQ ID NO:31), LCTPSA (SEQ ID NO:32), ICTPAA (SEQ ID NO:33), MCTPSA (SEQ ID NO:34), VCTPSA (SEQ ID NO:35), LCSPSA (SEQ ID NO:36), LCAPSA (SEQ ID NO:37), LCVPSA ​​(SEQ ID NO:38), and LCGPSA (SEQ ID NO:39).

[0275] fGly-containing sequence In general, the FGE used to promote the conversion of a cysteine ​​or serine to fGly in the sulfatase motif of the aldehyde tag of the target polypeptide is selected according to the sulfatase motif present in the aldehyde tag. The FGE may be native to the host cell in which the aldehyde-tagged 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-tagged protein in a human cell expressing FGE, or in a host cell, usually a mammalian cell, genetically modified to express human FGE. In general, FGE suitable for use in generating fGly-modified antibodies can be obtained from a naturally occurring source or can be synthetically produced. For example, a suitable FGE can be derived from a biological source that naturally produces FGE or is genetically modified to express a recombinant gene encoding FGE. Nucleic acids encoding several FGEs are known and readily known in the art.

[0276] After the action of FGE on the sulfatase motif, Z 1 is oxidized to generate a 2-formylglycine (fGly) residue. Furthermore, after both the FGE-mediated conversion and reaction with a reactive partner containing the moiety of interest, Z in the above formula is 1 The fGly position in is covalently attached to a moiety of interest (e.g., a detectable label, a water soluble polymer, a polypeptide, a drug, an active agent, etc.). Thus, the present disclosure provides antibodies having an amino acid sequence modified to include an fGly moiety.

[0277] Upon action of FGE on the antibody heavy and / or light chain, the serine or cysteine ​​in the sulfatase motif is modified to fGly. Thus, the fGly-containing sulfatase motif can be of the formula: X 1 (fGly)X 2 Z 2 X 3 Z 3(SEQ ID NO:40) (I''') During the ceremony, fGly is a formylglycine residue, Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0278] As described above, to produce a conjugate, a polypeptide containing an fGly residue may be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety of a linker attached to the drug or active agent (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety, as described above) to produce an fGly'-containing sulfatase motif. As used herein, the term fGly' refers to the amino acid residue of the sulfatase motif that is coupled to a drug or active agent through a linker as described herein. Thus, the present disclosure provides antibody conjugates.

[0279] In certain embodiments, the antibody conjugate comprises a fGly'-containing sulfatase motif of the following formula: X 1 (fGly')X 2 Z 2 X 3 Z 3 (SEQ ID NO: 41) (II) During the ceremony, fGly' is an amino acid residue coupled to a drug or active agent through a linker described herein; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0280] In certain embodiments, the sequence of formula (II) is located at the C-terminus of the heavy chain constant region of the antibody. In some cases, the heavy chain constant region comprises a sequence of formula (II): X1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue coupled to a drug or active agent through a linker described herein; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., 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; This sequence is C-terminal to the amino acid sequence QKSLSLSPGK, and this sequence can include 1, 2, 3, 4, 5, or 5-10 amino acids that are not present in the native wild-type heavy chain Ig constant region.

[0281] In a specific embodiment, the heavy chain constant region comprises the sequence SLSLSPGSL(fGly')TPSRGS (SEQ ID NO:42) at the C-terminus of an Ig heavy chain, for example, instead of the native SLSLSPGK (SEQ ID NO:43) sequence.

[0282] In certain embodiments, the amino acid residue (fGly') coupled to the drug or active agent is located in the light chain constant region of the antibody. In certain embodiments, the light chain constant region comprises the sequence of formula (II): X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue coupled to a drug or active agent through a linker described herein; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., 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; This sequence is C-terminal to the amino acid sequence KVDNAL (SEQ ID NO:44) and / or N-terminal to the amino acid sequence QSGNSQ (SEQ ID NO:45).

[0283] In a specific embodiment, the light chain constant region comprises the sequence KVDNAL(fGly')TPSRQSGNSQ (SEQ ID NO:46).

[0284] In certain embodiments, the amino acid residue (fGly') coupled to the drug or active agent is located in the heavy chain CH1 region of the antibody. In certain embodiments, the heavy chain CH1 region comprises the sequence of formula (II): X 1 (fGly')X 2 Z 2 X 3 Z 3 (II) During the ceremony, fGly' is an amino acid residue coupled to a drug or active agent through a linker described herein; Z 2 is either a proline or an alanine residue (which can also be represented by (P / A)), Z 3 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 Provided that there exists X 2 and X 3 can be independently any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., 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; This sequence is C-terminal to the amino acid sequence SWNSGA (SEQ ID NO:47) and / or N-terminal to the amino acid sequence GVHTFP (SEQ ID NO:48).

[0285] In a specific embodiment, the heavy chain CH1 region comprises the sequence SWNSGAL(fGly')TPSRGVHTFP (SEQ ID NO:49).

[0286] Figure 16A depicts a site map showing potential modification sites for the generation 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: 1) showing potential modification sites in the Ig light chain, and the lower sequence is the amino acid sequence of a conserved region of an Ig heavy chain polypeptide (SEQ ID NO: 2) (GenBank Accession No. AAG00909) showing potential modification sites in the Ig heavy chain. The numbering of the heavy and light chains is based on the full-length heavy and light chains.

[0287] 16B-16C depict alignments of Homo sapiens immunoglobulin heavy chain constant regions of IgG1 (SEQ ID NO:3, GenBank P01857.1), IgG2 (SEQ ID NO:4, GenBank P01859.2), IgG3 (SEQ ID NO:5, GenBank P01860.2), IgG4 (SEQ ID NO:6, GenBank AAB59394.1), and IgA (SEQ ID NO:7, GenBank AAAT74070), showing modification sites where aldehyde tags may be provided in the immunoglobulin heavy chains. The numbering of the heavy and light chains is based on the total heavy and light chains.

[0288] Figure 16D depicts an alignment of immunoglobulin light chain constant regions and indicates modification sites where aldehyde tags can be provided in the immunoglobulin light chain. Sequence 1 = Homo sapiens kappa light chain constant region, GenBank CAA75031.1, SEQ ID NO: 8. Sequence 2 = Homo sapiens kappa light chain constant region, GenBank BAC0168.1, SEQ ID NO: 9. Sequence 3 = Homo sapiens lambda light chain constant region, GenBank CAA75033, SEQ ID NO: 10. Sequence 4 = Mus musculus light chain constant region, GenBank AAB09710.1, SEQ ID NO: 11. Sequence 5 = Rattus norvegicus light chain constant region, GenBank AAD10133, SEQ ID NO: 12.

[0289] In some embodiments, the sulfatase motif is at a position other than, or in addition to, the C-terminus of an Ig polypeptide heavy chain. An isolated aldehyde-tagged polypeptide can comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described herein, wherein the sulfatase motif is within or adjacent to a surface-accessible loop region of the polypeptide heavy chain constant region.

[0290] Exemplary surface accessible loop regions of IgG1 heavy chains include: 1) ASTKGP, 2) KSTSGGT, 3) PEPV, 4) NSGALTSG, 5) NSGALTSGVHTFPAVLQSSGL, 6) QSSGL, 7) VTV, 8) QTY, 9) TQTY, 10) HKPSN, 11) EPKSCDKTHTCPPCPAPELLGG, 12) FPPKP, 13) ISRTP, 14) DVSHEDPEV, 15) NSGALTSGVHTFPAVLQSSGL, 16) NSGALTSGVHTFPAVLQSSGL, 17) NSGALTSGVHTFPAVLQSSGL, 18) NSGALTSGVHTFPAVLQSSGL, 19) NSGALTSGVHTFPAVLQSSGL, 20) NSGALTSGVHTFPAVLQSSGL, 21) NSGALTSGVHTFPAVLQSSGL, 22) NSGALTSGVHTFPAVLQSSGL, 23) NSGALTSGVHTFPAVLQSSGL, 24) NSGALTSGVHTFPAVLQSSGL, 25) NSGALTSGVHTFPAVLQSSGL, 26) NSGALTSGVHTFPAVLQSSGL, 27) NSGALTSGVHTFPAVLQSSGL, 28) NSGALTSGVHTFPAVLQSSGL, 29) NSGALTSGVHTFPAVLQSSGL, 30) NSGALTSGVHTFPAVLQSSGL, 31) NSGALTSGVHTFPAVLQSSGL, 32) NSGALTSGVHTFPAVLQSSGL, 33) NSGALTSGVHTFPAVLQSSGL, 34) NSGALTSGVHTFPAVLQSSGL, 35) NS ) SHEDPEV, 16) DG, 17) DGVEVHNAK, 18) HNA, 19) QYNST, 20) VLTVL, 21) GKE, 22) NKALPAP, 23) SKAKGQPRE, 24) KAKGQPR, 25) PPSRKELTKN, 26) YPSDI, 27) NGQPENN, 28) TPPVLDSDGS, 29) HEALHNHYTQKSLSLSPGK, and 30) SLSPGK.

[0291] Exemplary surface accessible loop regions of IgG2 heavy chains include: 1) ASTKGP, 2) PCSRSTSESTAA, 3) FPEPV, 4) SGALTSGVHTFP, 5) QSSGLY, 6) VTV, 7) TQT, 8) HKP, 9) DK, 10) VAGPS, 11) FPPKP, 12) RTP, 13) DVSHEDPEV, 14) DGVEVHNAK, 15) FN, 16) VLTVV, 17) GKE, 18) NKGLPAP, 19) SKTKGQPRE, 20) PPS, 21) MTKNQ, 22) YPSDI, 23) NGQPENN, 24) TPPMLDSDGS, 25) GNVF, and 26) HEALHNHYTQKSLSLSPGK.

[0292] Exemplary surface accessible loop regions of IgG3 heavy chains include: 1) ASTKGP, 2) PCSRSTSGGT, 3) FPEPV, 4) SGALTSGVHTFPAVLQSSG, 5) V, 6) TQT, 7) HKPSN, 8) RVELKTPLGD, 9) CPRCPKP, 10) PKSCDTPPPCPRCPAPELLGG, 11) FPPKP, 12) RTP, 13) DVSHEDPEV, 14) DGVEVHNAK, 15) YN, 16) VL, 17) GKE, 18) NKALPAP, 19) SKTKGQPRE, 20) PPSREEMTKN, 21) YPSDI, 22) SSGQPENN, 23) TPPMLDSDGS, 24) GNI, 25) HEALHNR, and 26) SLSPGK.

[0293] Exemplary surface accessible loop regions of IgG4 heavy chains include: 1) STKGP, 2) PCSRSTSESTAA, 3) FPEPV, 4) SGALTSGVHTFP, 5) QSSGLY, 6) VTV, 7) TKT, 8) HKP, 9) DK, 10) YG, 11) CPAPEFLGGPS, 12) FPPKP, 13) RTP, 14) DVSQEDPEV, 15) DGVEVHNAK, 16) FN, 17) VL, 18) GKE, 19) NKGLPSS, 20) SKAKGQPREP, 21) PPSQEEMTKN, 22) YPSDI, 23) NG, 24) NN, 25) TPPVLDSDGS, 26) GNVF, and 27) HEALHNHYTQKSLSLSLGK.

[0294] Exemplary surface accessible loop regions of IgA heavy chains include: 1) ASPTSPKVFPLSL, 2) QPDGN, 3) VQGFFPQEPL, 4) SGQGVTARNFP, 5) SGDLYTT, 6) PATQ, 7) GKS, 8) YT, 9) CHP, 10) HRPA, 11) LLGSE, 12) GLRDASGV, 13) SSGKSAVQGP, 14) GCYS, 15) CAEP, 16) PE, 17) SGNTFRPEVHLLPPPSEELALNEL, 18) ARGFS, 19) QGSQELPREKY, 20) AV, 21) AAED, 22) HEAL, and 23) IDRLAGKPTHVNVSVVMAEVDGTCY.

[0295] Exemplary surface-accessible loop regions of an Ig light chain (e.g., a human kappa light chain) include: 1) RTVAAP, 2) PPS, 3) Gly (see, e.g., the Gly at position 150 of the human kappa light chain sequence depicted in Figure 8C), 4) YPREA, 5) PREA, 6) DNALQSGN, 7) TEQDSKDST, 8) HK, 9) HQGLSS, and 10) RGEC.

[0296] Exemplary surface accessible loop regions of an Ig lambda light chain include QPKAAP, PPS, NK, DFYPGAV, DSPPVKAG, TTP, SN, HKS, EG, and APTECS.

[0297] The constant region of the HC of the antibodies disclosed herein may be selected from one of the following sequences:

[0298] CT tagged (aldehyde tag - bold) (Sequence Table 1) TIFF2024529466000029.tif47159

[0299] In the above sequence, the italicized residues at the C-terminus of the heavy chain constant region replace the lysine residue at the C-terminus of a standard IgG1 heavy chain. The bolded residues (LCTPSR) among the italicized residues constitute an aldehyde tag, where C is converted to a fGly residue by FGE upon expression of the heavy chain. The fGly can be converted to fGly', where fGly' refers to the amino acid residue of the antibody that is coupled to the moiety of interest (e.g., a drug). The non-bolded residues among the italicized residues are additional residues that differ from the standard IgG1 heavy chain sequence.

[0300] 58Q-1 (aldehyde tag - bold and replacement of "EEM" with "DEL") (Sequence Table 2) TIFF2024529466000030.tif47158

[0301] 61G-1 (aldehyde tag - bold and replacement of "EEM" with "DEL") (Sequence Table 3) TIFF2024529466000031.tif47158

[0302] 91N-1 (aldehyde tag - bold and replacement of "EEM" with "DEL") (Sequence Table 4) TIFF2024529466000032.tif45158

[0303] 116E-1 (aldehyde tag - bold and replacement of "EEM" with "DEL") (Sequence Table 5) TIFF2024529466000033.tif46157

[0304] 58Q-2 (aldehyde tag-bold) (Sequence Table 6) TIFF2024529466000034.tif47158

[0305] 61G-2 (aldehyde tag-bold) (Sequence Table 7) TIFF2024529466000035.tif46159

[0306] 91N-2 (aldehyde tag - bold) (Sequence Table 8) TIFF2024529466000036.tif46158

[0307] 116E-2 (aldehyde tag-bold) (Sequence Table 9) TIFF2024529466000037.tif46157

[0308] 58Q-3 (aldehyde tag - bold, "KKV" replaced with "KRV" and "EEM" replaced with "DEL") (Sequence Table 10) TIFF2024529466000038.tif48159

[0309] 61G-3 (aldehyde tag - bold, "KKV" replaced with "KRV" and "EEM" replaced with "DEL") (Sequence Table 11) TIFF2024529466000039.tif47160

[0310] 91N-3 (aldehyde tag - bold, "KKV" replaced with "KRV" and "EEM" replaced with "DEL") (Sequence Table 12) TIFF2024529466000040.tif46159

[0311] 116E-3 (aldehyde tag - bold, "KKV" replaced with "KRV" and "EEM" replaced with "DEL") (Sequence Table 13) TIFF2024529466000041.tif47159

[0312] 58Q-4 (aldehyde tag - bold and replacement of "KKV" with "KRV") (Sequence Table 14) TIFF2024529466000042.tif47159

[0313] 61G-4 (aldehyde tag - bold and replacement of "KKV" with "KRV") (Sequence Table 15) TIFF2024529466000043.tif46159

[0314] 91N-4 (aldehyde tag - bold and replacement of "KKV" with "KRV") (Sequence Table 16) TIFF2024529466000044.tif47160

[0315] 116E-4 (aldehyde tag - bold and replacement of "KKV" with "KRV") (Sequence Table 17) TIFF2024529466000045.tif47159

[0316] The bolded residues (LCTPSR) constitute the aldehyde tag, where C is converted to a fGly residue by FGE upon expression of the heavy chain. fGly can be converted to fGly', where fGly' refers to the amino acid residue of the antibody that is coupled to the moiety of interest (e.g., a drug).

[0317] Drugs In some cases, the antibodies of the present disclosure may comprise a drug covalently attached to the heavy and / or light chain of the antibody (e.g., the W-group in the conjugates of Formula (I) and compounds of Formula (III) described herein). 1 For example, the antibody conjugates of the present disclosure have a substituent W 1 The conjugated antibody may include a drug or active agent as a reactive partner. Any of a number of drugs are suitable for use, or can be modified to be suitable for use, as a reactive partner for conjugation to an antibody. "Drug" includes small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), and the like.

[0318] "Small molecule drug" as used herein refers to a compound, e.g., an organic compound, that exhibits a desired pharmacological activity and is generally of molecular weight of about 800 Da or less, or 2000 Da or less, but can include molecules up to 5 kDa and can be as large as about 10 kDa. 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.

[0319] In certain embodiments, the drug or active agent may be maytansine. "Maytansine," "maytansine moiety," "maytansine active drug moiety," and "maytansinoid" refer to maytansine and its analogues and derivatives, and pharma- ceutical active maytansine moieties and / or portions thereof. The maytansine conjugated to the polypeptide may be any of a variety of maytansinoid moieties, including, but not limited to, maytansine and its analogues and derivatives described herein.

[0320] In certain embodiments, the drug or active agent may be an auristatin, or an analog or derivative thereof, or a pharma- ceutically active auristatin moiety and / or moiety thereof. The auristatin conjugated to the polypeptide may be any of the various auristatin moieties, including, but not limited to, the auristatins and analogs and derivatives thereof described herein. Examples of drugs used in the conjugates and compounds described herein include, but are not limited to, auristatins or auristatin derivatives, such as monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and derivatives thereof. In certain embodiments, the drug is MMAE.

[0321] In certain embodiments, the drug or active agent may be a duocarmycin, or an analog or derivative thereof, or a pharma- ceutically active duocarmycin moiety and / or moiety thereof. The duocarmycin conjugated to the polypeptide may be any of the various duocarmycin moieties, including, but not limited to, the duocarmycins and analogs and derivatives thereof described herein. Examples of drugs used in the conjugates and compounds described herein include, but are not limited to, duocarmycins, or duocarmycin derivatives, such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof. In some embodiments, the duocarmycin is a duocarmycin analog, including, but not limited to, adozelesin, bizelesin, or carzelesin.

[0322] In certain embodiments, the drug or active agent may be a topoisomerase inhibitor, such as camptothecin or an analog or derivative thereof, or a pharma- ceutically active camptothecin moiety and / or portion thereof. The camptothecin conjugated to the subject antibody may be any of the various camptothecin moieties, such as, but not limited to, camptothecin and its analogs and derivatives, described in U.S. Application No. 17 / 575,481, filed Jan. 13, 2022, the disclosure of which is incorporated herein by reference. Additional examples of topoisomerase inhibitors for use in the conjugates described herein include, but are not limited to, camptothecin or camptothecin derivatives, such as SN-38, belotecan, exatecan, 9-aminocamptothecin (9-AC), and derivatives thereof.

[0323] In certain embodiments, the drug is selected from a cytotoxin, a kinase inhibitor, an immunostimulant, a Toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.

[0324] For example, a cytotoxin can include any compound that leads to cell death (eg, necrosis or apoptosis) or reduced cell survival.

[0325] Kinase inhibitors include, but are not limited to, adavosertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitinib, tucatinib, vandetanib, vemurafenib, and the like.

[0326] Immunostimulants include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, etc. TLR agonists include, but are not limited to, imiquimod, resiquimod, etc.

[0327] Oligonucleotide drugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, voranesorsen, inotersen, tofersen, tominersen, and the like.

[0328] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like.

[0329] Cytokines include albinterferon alfa-2B, aldesleukin, ALT-801, anakinra, ancestim, avotermin, valgrastim, bempegaldesleukin, binetrakin, syntredekin besudotox, CTCE-0214, darbepoetin alfa, denileukin diftitox, dulanermin, eddekin alfa, emfilamine, epoetin delta, erythropoietin, human interleukin-2, interferon alfa, interferon alfa-2c, interferon alfa-n1, interferon alfa-n3, interferon alfacon-1, interferon beta-1a, interferon beta-1b, interferon gamma-1b, interferon kappa. These include, but are not limited to, interleukin-1 alpha, interleukin-10, interleukin-7, lenograstim, religistim, lipegfilgrastim, lorcafusp alfa, Maxy-G34, methoxypolyethylene glycol epoetin beta, molgramostim, muprestim, nagressipene, oprelvekin, pegfilgrastim, pegilodecakin, peginterferon alpha-2a, peginterferon alpha-2b, peginterferon beta-1a, peginterferon lambda-1a, recombinant CD40 ligand, regramostim, romiplostim, sargramostim, thrombopoietin, tucotuzumab celmoleukin, viral macrophage inflammatory protein, and the like.

[0330] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.

[0331] "Peptide drug" as used herein refers to an amino acid-containing polymeric compound and is intended to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, as well as proteins, and peptidomimetics. Peptide drugs can be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). Peptide drugs can range in molecular weight, from 200 Da to 10 kDa or more. Suitable peptides include, but are not limited to, cytotoxic peptides, angiogenic peptides, antiangiogenic peptides, peptides that activate B cells, peptides that activate T cells, antiviral peptides, peptides that inhibit viral fusion, peptides that increase the production of one or more lymphocyte populations, antimicrobial peptides, growth factors, growth hormone releasing factors, vasoactive peptides, anti-inflammatory peptides, peptides that regulate glucose metabolism, antithrombotic peptides, antinociceptive peptides, vasodilatory peptides, platelet aggregation inhibitors, analgesics, and the like.

[0332] Additional examples of drugs for use in the conjugates and compounds described herein include, but are not limited to, tubulysin M, calicheamicin, STAT3 inhibitors, alpha-amanitin, aurora kinase inhibitors, belotecan, and anthracyclines.

[0333] In some cases, the drug is a toxin, for example, a cytotoxin.Ribosome-inactivating protein (RIP), a class of proteins that are ubiquitous in higher plants, is an example of such a cytotoxin.Suitable cytotoxins include, but are not limited to, ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pokeweed antiviral protein (PAP), botulinum toxin, bryodin, momordin, and buganin, etc.

[0334] In some cases, the drug is a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (e.g., non-proteinaceous) compounds that reduce the proliferation of cancer cells, including cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, or steroid hormones. Peptide compounds can also be used.

[0335] Suitable cancer chemotherapeutic agents include dolastatins and their active analogs and derivatives, and auristatins and their active analogs and derivatives. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives, and duocarmycins and their active analogs and derivatives.

[0336] Drugs that act to reduce cell proliferation are known in the art and are widely used.Such drugs include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including but not limited to mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0337] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0338] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines, daunorubicin hydrochloride, and the like. These include, but are not limited to, cyclosporines (daunomycin, rubidomycin, cerubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives; phenoxyzombicyclopeptides, such as dactinomycin; basic glycopeptides, such as bleomycin; anthraquinone glycosides, such as plicamycin (mithramycin); anthracenediones, such as mitoxantrone; azirinopyrroloindole diones, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like.

[0339] Other antiproliferative cytotoxic agents are navelbine, CPT-11, anastrozole, letrozole, capecitabine, reloxaphene, cyclophosphamide, ifosfamide, and droloxifene.

[0340] Microtubule acting agents with antiproliferative activity are also suitable for use, including, but not limited to, allocolchicine (NSC406042), halichondrin B (NSC609395), colchicine (NSC757), colchicine derivatives (e.g., NSC33410), dolstatin 10 (NSC376128), maytansine (NSC153858), rhizoxin (NSC332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including, but not limited to, epothilone A, epothilone B, discodermolide, estramustine, nocodazole, and the like.

[0341] Hormonal regulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, corticosteroids such as prednisone, dexamethasone, and the like; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; and corticosteroids such as aminoglutethimide; 17α-ethynyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogen stimulates proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this activity.

[0342] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epiphyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), and the like.

[0343] Taxanes are suitable for use. "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) is readily prepared utilizing techniques known to those skilled in the art (WO 94 / 078). 82, 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 from a variety of commercial sources including, for example, Sigma Chemical Co. St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0344] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogues and derivatives (e.g., TAXOTERE™ docetaxel, as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0345] Also included within the term "taxane" are various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives, piperazino and piperazino derivatives.

[0346] Embodiments of the disclosure include conjugates in which an antibody is conjugated to two or more drug moieties, e.g., 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 moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugate has an average drug-to-antibody ratio (DAR) (molar ratio) in the range of 0.1-20, or 0.5-20, or 1-20, e.g., 1-19, or 1-18, or 1-17, or 1-16, or 1-15, or 1-14, or 1-13, or 1-12, or 1-11, or 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2. In certain embodiments, the conjugate has an average DAR of 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugate has an average DAR of 1-10. In certain embodiments, the conjugate has an average DAR of 1-5. In certain embodiments, the conjugate has an average DAR of 5-10. By average is meant the arithmetic mean.

[0347] Drugs conjugated to polypeptides can be modified to incorporate reactive partners for reaction with the polypeptide. When the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be located at the N-terminal region, N-terminus, C-terminal region, C-terminus, or at an internal position of the peptide. For example, one method involves synthesizing a peptide drug with an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide can be reacted with a compound containing a carboxylic acid group and an oxy-N-Boc group. As an example, the amino group of the peptide is reacted with 3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on the compound containing a carboxylic acid group and an oxy-N-protecting group can include different numbers of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound containing a carboxylic acid group and an oxy-N-protecting group occurs through standard peptide coupling chemistry.Examples of peptide coupling reagents that can be used include DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluorene), oroformamidinium hexafluorophosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium 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-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), BOP-Cl (Bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(tris(2-oxo-3-oxazolidinyl)phosphinic acid chloride) (pyrrolidino)phosphonium tetrafluorophosphate), BrOP (bromo tris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), PyBrOP (bromo tris(pyrrolidino)phosphonium hexafluorophosphate). As non-limiting examples, HOBt and DIC can be used as peptide coupling reagents.

[0348] Deprotection to expose aminooxy functionality is carried out on peptides containing N-protecting groups. Deprotection of N-oxysuccinimide groups occurs, for example, according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al. Specific deprotection conditions include hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of Boc protecting groups occurs with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. Products and intermediates can be purified by conventional means, such as HPLC purification.

[0349] Those skilled in the art will understand that factors such as pH and steric hindrance (e.g., proximity of amino acid residues to reaction with the intended reactive partner) are important. Modifying reaction conditions to provide optimal conjugation conditions is well within the skill of the art and routine in the art. When conjugation is performed with polypeptides present in or on living cells, conditions are selected to be physiologically compatible. For example, the pH may be temporarily lowered for a time sufficient to allow the reaction to occur but within a period tolerated by the cells (e.g., about 30 minutes to 1 hour). Physiological conditions for performing modification of polypeptides on cell surfaces may be similar to those used in ketone-azide reactions in the modification of cells with cell surface azides (see, e.g., US 6,570,040).

[0350] Small molecule compounds that contain or are modified to contain an α-nucleophilic group that functions as a reactive partner with the compounds or conjugates disclosed herein are also contemplated for use as drugs in the polypeptide drug conjugates of the present disclosure.General methods are known in the art for chemical synthesis schemes and conditions useful for synthesizing the compounds of interest (see, for example, 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).

[0351] Methods for Producing Antibodies The subject antibodies can be produced by any known method, such as conventional synthetic methods for protein synthesis, recombinant DNA methods, and the like.

[0352] If the subject antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When a polypeptide is chemically synthesized, synthesis can proceed via liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is bound to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is an example of a suitable method for chemically synthesizing the subject antibodies. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the subject antibodies.

[0353] Standard recombinant methods can be used to produce the subject antibodies. For example, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. 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 sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vectors are incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and collection and purification of the antibody.

[0354] Due to the degeneracy of the code, a variety of nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be produced by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of a previously prepared variant of the desired polynucleotide.

[0355] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to permit detection of cells transformed with the desired DNA sequence.

[0356] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding an antibody of interest. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells.

[0357] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce the polypeptides (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the present 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 can include expression control sequences such as replication origins, promoters, and enhancers, as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like.

[0358] Once synthesized (either chemically or recombinantly), whole antibodies, dimers thereof, 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, and the like (see generally, Scopes, Protein Purification, Springer-Verlag, NY, (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, macromolecules other than the subject antibodies.

[0359] composition The conjugates of the present disclosure can be formulated in a variety of different ways. Generally, when the conjugate is a polypeptide drug conjugate (e.g., an antibody drug conjugate), the conjugate is formulated in a manner that is compatible with the drug conjugated to the polypeptide, the condition being treated, and the route of administration being used.

[0360] In some embodiments, a pharmaceutical composition is provided comprising any of the conjugates of the present disclosure and a pharma- ceutically acceptable excipient.

[0361] The conjugates (e.g., antibody-drug conjugates) can be provided in any suitable form, for example, in the form of a pharma- ceutically acceptable salt, and can be formulated for any suitable route of administration, for example, oral, topical, or parenteral. When the conjugates are provided as injectable liquids (such as in embodiments in which they are administered intravenously or directly to a tissue), the conjugates can be provided as ready-to-use dosage forms or as reconstitutable storage-stable powders or liquids comprised of pharma- ceutically acceptable carriers and excipients.

[0362] Methods for formulating the conjugates can be adapted from those readily available. For example, the conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharma- ceutically acceptable carrier (e.g., saline). The pharmaceutical composition can optionally include other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to a mammal, e.g., suitable for administration to a human.

[0363] For example, the present disclosure provides compositions comprising the subject antibody conjugates. The subject antibody conjugate compositions can include, in addition to the subject antibody conjugates, one or more of the following: a salt, e.g., NaCl, MgCl2, KCl, MgSO4, etc., a buffer, e.g., 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), etc., a solubilizer, a detergent, e.g., a non-ionic detergent, e.g., Tween-20, etc., a protease inhibitor, glycerol, etc.

[0364] In certain embodiments, the present disclosure provides compositions, including pharmaceutical compositions, that include the subject antibody conjugates. In general, the formulations include an effective amount of the subject antibody conjugates. By "effective amount" is meant a dosage sufficient to produce a desired result, e.g., a reduction in the number of cancerous cells. In some cases, the desired result is at least a reduction in the symptoms of a malignant tumor compared to a control.

[0365] formulation In the subject method, the subject antibody conjugate can be administered to a host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Thus, the antibody conjugate can be incorporated into various formulations for therapeutic administration. More specifically, the subject antibody conjugate can be formulated into a pharmaceutical composition by combining with a suitable pharma-ceutically acceptable carrier or diluent, and can be formulated into a solid, semi-solid, liquid, or gaseous form of preparation, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0366] In pharmaceutical dosage forms, the subject antibody conjugates can be administered in the form of their pharma- ceutically acceptable salts, or they can also be used alone or in suitable association, as well as in combination with other pharma- ceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0367] For oral formulations, the subject antibody conjugates can be used alone or in combination with suitable excipients for making tablets, powders, granules, or capsules, e.g., conventional excipients such as lactose, mannitol, corn starch, or potato starch, binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin, disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose, lubricants such as talc or magnesium stearate, and, if desired, diluents, buffers, humectants, preservatives, and flavorings.

[0368] The subject antibody conjugates can be formulated into preparations for injection by dissolving, suspending, or emulsifying the preparation in aqueous or non-aqueous solvents, such as vegetable 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, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0369] Pharmaceutical compositions containing the subject antibody conjugates are prepared by mixing the antibody conjugates having the desired purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or isotonicity agents. Acceptable carriers, excipients and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, asparagine, isoleucine, arginine, glyc ... , amino acids such as leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronic, Triton-X, or polyethylene glycol (PEG).

[0370] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with the lyophilized preparation being reconstituted with a sterile solution prior to administration.The standard procedure for reconstituting a lyophilized composition is to add back a volume of purified water (typically equal to the volume removed during lyophilization), although solutions containing antimicrobial agents may be used to produce pharmaceutical compositions for parenteral administration.

[0371] Exemplary antibody conjugate concentrations in a subject pharmaceutical composition can range from about 1 mg / mL to about 200 mg / ml, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0372] Aqueous formulations of antibody conjugates can be prepared in pH buffer solutions at a pH ranging from, for example, about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0373] Cryoprotectants can be added to protect labile active ingredients (e.g., proteins) against destabilizing conditions during the lyophilization process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants can be included in amounts of about 10 nM to 500 nM.

[0374] In some embodiments, a subject formulation comprises a subject antibody conjugate and one or more agents (e.g., surfactants, buffers, stabilizers, isotonicity agents), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof, hi other embodiments, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0375] For example, a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration and can contain from about 1 mg / mL to about 200 mg / mL of a subject antibody conjugate, from about 0.001% to about 1% of at least one surfactant, from about 1 mM to about 100 mM of a buffer, optionally from about 10 mM to about 500 mM of a stabilizer, and from about 5 mM to about 305 mM of an isotonicity agent, and has a pH of from about 4.0 to about 7.0.

[0376] As another example, a subject parenteral formulation is a liquid or lyophilized formulation comprising from about 1 mg / mL to about 200 mg / mL of a subject antibody conjugate, 0.04% Tween 20 w / v, 20 mM L-histidine, and 250 mM sucrose, and has a pH of 5.5.

[0377] 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 an antibody conjugate of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharma- ceutically acceptable diluent, carrier, or vehicle. The specifications for 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.

[0378] The subject antibody conjugates can be administered as injectable preparations. Typically, injectable compositions are prepared as liquid solutions or suspensions, and solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. Preparations can also be emulsified, or the antibody conjugates can be encapsulated in liposome vehicles.

[0379] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharma- ceutically 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.

[0380] In some embodiments, the subject antibody conjugates are formulated in controlled release formulations. Sustained release preparations can be prepared using methods well known in the art. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody conjugates, where 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 acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Possible loss of biological activity and possible changes in immunogenicity of the antibodies contained in the sustained release preparations can be prevented by using appropriate additives, by controlling the moisture content, and by developing specific polymer matrix compositions.

[0381] Physical systems include, but are not limited to, reservoir systems with rate-controlling membranes such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlling membranes such as hollow fibers, ultra-microporous cellulose triacetate, and porous polymer matrices and foams; systems physically dissolved in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, erodible, environmental agent-permeable, and degradable); and monolithic systems comprising materials physically dispersed in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, erodible, environmental agent-permeable, and degradable); laminated structures comprising a reservoir layer that is chemically similar or different from an outer controlling layer; and other physical methods such as osmotic pumps or adsorption onto ion exchange resins.

[0382] Chemical systems include, but are not limited to, chemical erosion (eg, heterogeneous or homogeneous erosion) of a polymer matrix, or biological erosion (eg, heterogeneous or homogeneous) of a polymer matrix.

[0383] Dosage The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the size, body surface area, age of the patient, the specific compound being administered, the patient's sex, the time and route of administration, general health, and other drugs being administered concomitantly. The subject antibody conjugates can be administered in an amount of 1 ng / kg body weight to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight, although doses below or above this exemplary range are contemplated, particularly considering the aforementioned factors. If the regimen is a continuous infusion, this can also be within the range of 1 μg to 10 mg per kg body weight per minute.

[0384] One of skill in the art will readily understand that dosage levels can vary as a function of the particular antibody conjugate, the severity of the symptoms, and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0385] Route of administration The subject 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 local routes of administration.

[0386] Conventional pharma- ceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or adjusted, if desired, depending on the antibody conjugate and / or the desired effect. The subject antibody conjugate compositions may be administered in a single dose or multiple doses. In some embodiments, the subject antibody conjugate compositions are administered orally. In some embodiments, the subject antibody conjugate compositions are administered via the inhalation route. In some embodiments, the subject antibody conjugate compositions are administered intranasally. In some embodiments, the subject antibody conjugate compositions are administered topically. In some embodiments, the subject antibody conjugate compositions are administered intracranially. In some embodiments, the subject antibody conjugate compositions are administered intravenously.

[0387] The antibody conjugates can be administered to a host using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. In general, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0388] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrahepatic, and intravenous routes, e.g., any route of administration other than through the digestive tract. Parenteral administration can be carried out to effect systemic or local delivery of the subject antibodies. When systemic delivery is desired, administration typically involves invasive administration of pharmaceutical preparations, or topical administration that is systemically absorbed, or mucosal administration.

[0389] The subject antibody conjugates can also be delivered to a subject by enteral administration. Enteral administration routes include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[0390] Treatment means at least an amelioration of symptoms associated with a pathological condition from which the host suffers, where amelioration is used broadly to refer to at least a reduction in a parameter, e.g., the magnitude of a symptom, 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 the symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or arrested, e.g., terminated, such that the host is no longer afflicted by the pathological condition or at least the symptoms characterizing the pathological condition.

[0391] In some embodiments, the subject antibody conjugates are administered by injection, e.g., for systemic delivery (e.g., intravenous infusion) or to a localized site.

[0392] A variety of hosts (the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") can be treated according to the subject methods. Generally, such hosts are "mammals" or "mammals," terms used broadly to describe organisms within the mammalian family, 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 embodiments, the host will be a human.

[0393] Treatment method The present disclosure provides methods of treating malignancies, including solid tumors or hematological malignancies, which generally involve administering to an individual in need of treatment (e.g., an individual having a malignancy) an effective amount of a subject antibody conjugate, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy).

[0394] Malignant tumors include, for example, HCC, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, chronic lymphocytic leukemia, hairy cell leukemia, prolymphocytic leukemia, anal cancer, appendix cancer, bile duct cancer (e.g., cholangiocarcinoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, cancer of unknown primary site (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal 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, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer, and the like.

[0395] In some embodiments, an effective amount of a subject antibody conjugate is an amount that, when administered in one or more doses, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is 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, compared to the number of cancer cells in the individual in the absence of treatment with the antibody conjugate.

[0396] In some cases, the breast cancer is triple negative for estrogen, progesterone, and HER2. In some cases, the triple negative breast cancer is metastatic triple negative breast cancer. In some cases, the triple negative breast cancer is recurrent or refractory triple negative breast cancer. In some cases, the triple negative breast cancer is recurrent or refractory metastatic triple negative breast cancer.

[0397] Aspects of the present disclosure include a method of delivering a drug to a target site in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a conjugate according to the present disclosure, the administration being effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject.

[0398] In some embodiments, multiple doses of the antibody drug conjugate may be administered. The frequency of administration of the antibody drug conjugate may vary depending on any of a variety of factors, such as the severity of symptoms, the condition of the subject, etc. For example, in some embodiments, the antibody drug conjugate 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.

[0399] Combination therapy In some embodiments, the subject methods of treating malignancies involve administering a subject antibody conjugate and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, cancer chemotherapeutic agents (described above).

[0400] In some embodiments, the method of treatment may include administering to the subject a therapeutically effective amount of an immunomodulatory Therapeutic Agent. The immunomodulatory Therapeutic Agent may be an immune checkpoint inhibitor or an interleukin. The immune checkpoint inhibitor may inhibit A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT, or VISTA. The immune checkpoint inhibitor that inhibits PD-1 signaling may be an anti-PD-1 antibody. The anti-PD-1 antibody may be nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The immune checkpoint inhibitor that inhibits CTLA-4 may be an anti-CTLA-4 antibody. The anti-CTLA-4 antibody may be ipilimumab.

[0401] Suitable subjects for treatment A variety of subjects are suitable for treatment with the subject methods. Suitable subjects include any individual, e.g., a human, who has, has been diagnosed with, or has had a malignant tumor and is at risk for recurrence of the malignant tumor, has been treated for the malignant tumor with an agent other than the subject antibody conjugate (e.g., has been treated with a cancer chemotherapeutic agent) and has not responded to the agent, or has been treated for the malignant tumor with an agent other than the subject antibody conjugate (e.g., has been treated with a cancer chemotherapeutic agent) and has initially responded to the agent but subsequently become unresponsive (e.g., has relapsed).

[0402] Embodiment Particular embodiments of the present disclosure are described in the following enumerated clauses. These embodiments are exemplary only and are not intended to be limiting in scope. 1. A conjugate of formula (I): [ka] During the ceremony, Z is CR 4 or N, R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W 1 But it is a drug, W 2 is an antibody conjugate. 2. L, -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f - wherein a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is 1 to 6; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each independently represents a covalent bond, (C 12 ) Alkyl, Substituted (C1-C 12) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetals, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each m is an integer of 1 to 12; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 3. T 1 However, (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 Each independently represents a covalent bond, (C 12 ) Alkyl, Substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from the group consisting of -SO2-, and -P(O)OH-; (PEG) n but, [ka] wherein n is an integer from 1 to 30; EDA has the following structure: [ka] where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) [ka] and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The conjugate according to clause 2, wherein the groups may be cyclically linked to form a piperazinyl ring. 4. The conjugate according to clause 2 or 3, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. 5. The conjugate of clause 4, wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. 6. T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 is -CO-, T 3 However, (C1~C 12 ) alkyl, V 3 is -CO-, and d, e, and f are each 0; or T 1However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 But it doesn't exist. T 3 However, (PEG) n and V 3 is -CO-, and d, e, and f are each 0; or T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 But it doesn't exist. T 3 However, (PEG) n and V 3 is -CO-, T 4 However, (AA) p and V 4 But it doesn't exist. T 5 But PABC, V 5 does not exist, and f is 0, or T 1 However, (C1~C 12 ) alkyl, V 1 But -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 However, (AA) p and V 3 But it doesn't exist. T 4 But PABC, V 4 does not exist, and e and f are each 0, or T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T2 is an amino acid analogue, and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 However, (AA) p and V 4 But it doesn't exist. T 5 But PABC, V 5 does not exist, and The conjugate according to any of clauses 2 to 5, wherein f is 0. 7. The linker L has a structure selected from: [ka] TIFF2024529466000051.tif43154In formula, [ka] represents the bond of L to N in formula (I), * represents W 1 7. The conjugate according to any one of clauses 1 to 6, wherein L represents the bond to 8. The conjugate according to any one of clauses 1 to 7, wherein the drug is monomethyl auristatin E (MMAE). 9. A conjugate comprising: [ka] 9. The conjugate according to any one of clauses 1 to 8, selected from the group consisting of: TIFF2024529466000054.tif30158. 10. The conjugate according to any one of clauses 1 to 9, wherein the antibody is an IgG1 antibody. 11. The conjugate according to clause 10, wherein the antibody is an IgG1 kappa antibody. 12. The conjugate according to any one of clauses 1 to 11, wherein the antibody comprises a sequence having fGly', where fGly' is an amino acid residue coupled to the drug through a linker. 13. The conjugate according to any one of clauses 1 to 12, wherein the sequence is located at the C-terminus of the heavy chain constant region of the antibody. 14. The conjugate according to any one of clauses 1 to 12, wherein the sequence is located within the light chain constant region of an antibody. 15. The conjugate according to any one of clauses 1 to 12, wherein the sequence is located within the heavy chain CH1 region of an antibody. 16. The conjugate according to any one of clauses 1 to 12, wherein the sequence is located within the heavy chain CH2 region of an antibody. 17. The conjugate according to any one of clauses 1 to 12, wherein the sequence is located within the heavy chain CH3 region of an antibody. 18. A compound of formula (III), [ka] During the ceremony, Z is CR 4 or N, R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W 1 But it is a drug, a compound. 19. L, -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f - wherein a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is 1 to 6; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each independently represents a covalent bond, (C 12 ) Alkyl, Substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13OH) m -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetals, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each m is an integer of 1 to 12; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 20. T1 However, (C1~C 12 ) Alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 Each independently represents a covalent bond, (C 12 ) Alkyl, Substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from the group consisting of -SO2-, and -P(O)OH-; (PEG) n but, [ka] wherein n is an integer from 1 to 30; EDA has the following structure: [ka] where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) [ka] and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The compound according to clause 19, wherein the groups may be cyclically linked to form a piperazinyl ring. 21. The compound according to clause 19 or 20, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. 22. The compound according to clause 21, wherein the glycoside is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. twenty three. T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 is -CO-, T 3 However, (C1~C 12 ) alkyl, V 3 is -CO-, and d, e, and f are each 0; or T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 But it doesn't exist. T 3 However, (PEG) n and V 3 is -CO-, and d, e, and f are each 0; or T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 But, 4AP, V 2 But it doesn't exist. T 3 However, (PEG) n and V 3 is -CO-, T 4 is AA and V 4 does not exist, T 5 is PABC, and V 5 does not exist, and f is 0, or T 1 However, (C1~C 12 ) alkyl, V 1 But -CONH-, T 2 is (PEG) n and V 2 is -CO-, T 3 is AA and V 3 does not exist, T 4 is PABC, and V 4 does not exist, and e and f are each 0, or T 1 However, (C1~C 12 ) alkyl, V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist. T 5 But PABC, V 5does not exist, and 23. The compound according to any one of clauses 19 to 22, wherein f is 0. 24. The linker L has a structure selected from: [ka] TIFF2024529466000060.tif50156 [ka] represents the bond of L to N in formula (I), * represents W 1 24. The compound according to any one of clauses 18 to 23, wherein L represents the bond to 25. The compound according to any one of clauses 18 to 24, wherein the drug is MMAE. 26. A compound is [ka] 26. The compound according to any one of clauses 18 to 25, selected from the group consisting of: TIFF2024529466000063.tif29156. 27. A pharmaceutical composition comprising: A conjugate according to any one of clauses 1 to 17, and a pharma- ceutically acceptable excipient. 28. A method comprising: 18. A method comprising administering to a subject an effective amount of a conjugate according to any one of clauses 1 to 17. 29. A method of treating cancer in a subject, comprising: A method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of clause 27, wherein the administering is effective to treat cancer in the subject. 30. The method according to clause 29, wherein the cancer is breast cancer, ovarian cancer, lung cancer, or gastric cancer. 31. A method of delivering a drug to a target site in a subject, comprising: 28. A method comprising administering to a subject a pharmaceutical composition according to clause 27, wherein the administering is effective to deliver a therapeutically effective amount of the drug to a target site in the subject. EXAMPLES

[0403] The following examples are presented 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 following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp (base pair(s)), kb (kilobase(s)), pl (picoliter(s)), s or sec (second(s)), min (minute(s)), h or hr (hour(s)), aa (amino acid(s)), kb (kilobase(s)), bp (base pair(s)), nt (nucleotide(s)), im (intramuscular), ip (intraperitoneal), sc (subcutaneous), etc. Unless otherwise indicated, commercially available reagents referred to in the examples were used according to the manufacturer's instructions. The source of cells identified in the examples and throughout this specification by ECACC accession numbers is the European Collection of Cell Cultures (ECACC) (Salisbury, England). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 are not intended to be limiting in scope.

[0404] Example 1 Materials and Methods general Synthesis reagents were purchased from Sigma-Aldrich, Acros, AK Scientific, or other commercial sources and used without purification. Anhydrous solvents were obtained in sealed bottles from commercial sources. In all cases, solvents were removed under reduced pressure using a Buchi Rotovapor R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography system. Preparative HPLC purification was performed using a Waters preparative HPLC unit equipped with a Phenomenex Kinetex 5 μm EVO C18 150 × 21.2 mm column. HPLC analyses were performed on an Agilent 1100 Series analytical HPLC equipped with a Model G1322A degasser, Model G1311A quaternary pump, Model G1329A autosampler, Model G1314 variable wavelength detector, and an Agilent Poroshell 120SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid. HPLC was monitored at 254 or 205 nm. Low-resolution mass spectra (LRMS) were acquired on an Agilent Technology 6120 quadrupole LC / MS equipped with an Agilent 1260 Infinity HPLC system, a G1314 variable wavelength detector, and an Agilent Poroshell 120SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.1% formic acid.

[0405] Synthesis of MMAE constructs The structures of MMAE compounds 1-5 used in the study are shown below. Compound 1 was previously reported in Harpel et.al.Antibodies 2019,8,54. Compounds 2 and 3 were previously reported in Chuprakov et. al.Bioconjugate Chem.2021,32,4,746-754. Synthetic intermediates 6, 8, and 11 were obtained commercially from Shanghai Medicilon and used without purification. Monomethyl auristatin E9 was purchased from BroadPharm and used as received. [ka]

[0406] Synthesis of MMAE construct 4 Preparation of (9H-fluoren-9-yl)methyl 1,2-dimethyl-2-((1-(3-oxo-3-((2-(2-(3-oxo-3-(perfluorophenoxy)propoxy)ethoxy)ethyl)amino)propyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)hydrazine-1-carboxylate (7) [ka] In a 20 mL scintillation vial, compound 6 (56 mg, 87 μmol), pentafluorophenol (18 mg, 98 μmol), 35 μL DIPEA, and 1 mL EtOAc were combined. The resulting mixture was treated with 19 mg (122 μmol) EDC and stirred at room temperature for 3 days. The reaction mixture was diluted with DCM and washed with 1 M aqueous HCl, followed by saturated aqueous NaHCO3. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dried under high vacuum to give 59 mg of PFP-ester 7 (73 μmol, 84% yield) as a white solid, which was used further without purification.

[0407] LRMS(ESI):m / z 810.7[M+H] + , C 41 H 40Calculated value of m / z 810.8 for F5N5O7.

[0408] [ka] (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-amino-3-methylbutanamido)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- Preparation of 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-triyl triacetate (10) MMAE as the TFA salt (9, 150 mg, 0.18 mmol) and PNP carbonate 8 (160 mg, 0.16 mmol) were mixed in 2 mL of anhydrous DMF in an oven-dried 20 mL glass scintillation vial. This mixture was treated with 84 uL (0.48 mmol) of DIPEA and allowed to react for 2 h at room temperature. DIPEA was removed under vacuum and the remaining solution was treated with 32 uL (0.32 mmol) of piperidine at 0 °C for 7 h and then purified by reverse-phase preparative HPLC (C18, 5-95% gradient of acetonitrile-water with 0.05% TFA). Pure fractions were lyophilized to give 160 mg (0.12 mmol, 75% yield over two steps) of the title compound 10 as a white powder.

[0409] LRMS(ESI):m / z 1369.8[M+H] + , C 68 H 104 N8O 21 Calculated m / z 1369.7.

[0410] (2S,3S,4S,5R,6S)-6-(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- Preparation of 2,13-dioxa-4,7,10-triazatetradecyl)-2-((2S,5S)-19-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-isopropyl-2-methyl-4,7,17-trioxo-10,13-dioxa-3,6,16-triazanonadecanamido)phenoxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (4) In a glass vial, compound 10 (20 mg, 15 μmol) and PFP ester 7 (14 mg, 17 μmol) were combined in 0.5 mL of anhydrous DMF. The resulting mixture was treated with DIPEA (7 μL) and HOAt (1 mg) and stirred at room temperature for 1 h. The reaction mixture was concentrated under vacuum and reconstituted in 2.5 mL of MeOH. The solution was cooled to 0 °C, treated with 1.5 mL of 1 M aqueous LiOH, allowed to warm to room temperature and stirred for 2 h. The reaction mixture was neutralized by adding 1 M HCl, methanol was removed in vacuo, and the residue was purified by reverse-phase preparative HPLC (C18, 10-60% acetonitrile-water / 0.05% TFA). Pure fractions were combined and lyophilized to give 12 mg (7 μmol, 47% yield over two steps) of compound 4 as a white powder.

[0411] LRMS(ESI):m / z 1633.9[M+H] + , C 81 H 125 N 13 O 22 Calculated m / z 1633.9.

[0412] Synthesis of MMAE construct 5 Preparation of (R)-2-(3-(2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)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 (12) [ka] Carboxylic acid 11 (1.33 g, 1.67 mmol) was combined with pentafluorophenol (1.23 g, 6.68 mmol) in 6.5 mL of anhydrous DMF. This mixture was treated with EDCI-HCl (0.64 g, 3.34 mmol) in one portion at room temperature and stirred for 20 h until 11 was completely consumed as judged by HPLC analysis. The reaction mixture was directly purified by reverse-phase chromatography (C18 column, 0-80% acetonitrile-water with 0.05% TFA). Pure fractions were combined, concentrated under vacuum until cloudy, and lyophilized to give PFP-ester 12 (1.40 g, 1.46 mmol, 87% yield) as a tan powder.

[0413] LRMS(ESI):m / z 961.2[M+H] + , C 44 H 45 F5N6O 11 S calculated m / z 961.3.

[0414] (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropanamido) Preparation of (13)-2-(propyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxa-2,13-dioxa-4,7,10-triazatetrazyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate [ka] In a 20 mL glass vial, monomethyl auristatin A9 (720 mg, 1.0 mmol), 5 mL of anhydrous DMF, and 0.35 mL of DIPEA (2.0 mmol) were combined at room temperature. The resulting mixture was stirred and treated in portions with PNP carbonate 8 (1014 mg, 1.0 mmol) as a solid, followed by the addition of HOAt (136 mg, 1.0 mmol) in one portion at room temperature. The reaction mixture was stirred for 6 h until the reaction was judged complete (HPLC). The reaction mixture was poured into 30 mL of water, and the resulting precipitate was separated and collected by spinning, washed with 5 mL of water, and briefly dried under high vacuum to give 1.87 g of crude product 13 as a yellowish solid, which was carried on to the next step without purification.

[0415] Preparation of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)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-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (14) [ka] A solution of crude compound 13 (1.87 g) in 15 mL of THF was cooled to 0 °C in an ice bath and slowly treated with 1 M aqueous lithium hydroxide (3 mL). The reaction mixture was stirred at 0 °C for 3 h, then warmed to ambient temperature, treated with 3 mL of 1 M aqueous lithium hydroxide, and diluted with 3 mL of methanol. The resulting mixture was stirred at room temperature for 3 h until hydrolysis was complete (HPLC), then quenched by adding 1 M aqueous HCl to pH 7. The reaction mixture was then concentrated under reduced pressure and washed with 10 mL of MTBE. The aqueous layer was purified by reverse phase chromatography (C18 column, 0-40% acetonitrile-water with 0.05% TFA). The pure product fractions were combined, concentrated under reduced pressure, and lyophilized to give compound 14 as a white powder (735 mg, 0.60 mmol, 60% yield over two steps).

[0416] LRMS(ESI):m / z 1229.7[M+H] + , C 61 H 96 N8O 18 Calculated m / z 1229.7.

[0417] (2S,3S,4S,5R,6S)-6-(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 Preparation of 2-((2S,5S,18R)-22-(2-((1,2-dimethylhydrazine)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-tetraazadocosanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (5) [ka] To a stirred solution of compound 14 (735 mg, 0.60 mmol) in 3 mL of anhydrous DMA was added DIPEA (0.21 mL, 1.2 mmol) and a solution of PFP-ester 12 (575 mg, 0.60 mmol) in 2 mL of DMA at room temperature, followed by HOAt (84 mg, 0.60 mmol). The resulting mixture was stirred for 30 min until the coupling was judged complete (HPLC analysis) and then directly treated with 1.2 mL of piperidine at room temperature. After 15 min, the reaction mixture was purified by reverse phase chromatography (C18 column, 0-40% gradient of acetonitrile-water). Pure fractions were combined, concentrated under reduced pressure and room temperature, and then lyophilized to give compound 5 (808 mg, 0.45 mmol, 75% yield) as a white fluffy powder. LRMS (ESI): m / z 1783.9 [M+H] + , C 84 H 130 N 14 O 26 S calculated m / z 1783.9.

[0418] Example 2: Bioconjugation, purification, and HPLC analysis HIPS conjugation of aldehyde-tagged antibodies Antibodies (15 mg / mL) with one aldehyde tag were conjugated to the linker payload at 1.1 mM each. The reaction proceeded for 72 h at 37 °C in 20 mM sodium citrate, 50 mM NaCl at pH 5.5 containing 0.85-2.5% DMA (20 / 50 buffer). After conjugation, free drug was removed using a 30 kD MWCO 0.5 mL Amicon spin concentrator. The sample was added to the spin concentrator and centrifuged at 15,000 x g for 7 min, then diluted with 450 μL of 20 mM sodium citrate, 50 mM NaCl at pH 5.5, and centrifuged again. This process was repeated 10 times. To determine the DAR of the final product, the ADCs were investigated by analytical chromatography using HIC (Tosoh #14947) or PLRP-RP (Agilent PL1912-1802 1000A, 8um, 50x2.1mm) columns. HIC analysis used mobile phase A: 1.5M ammonium sulfate, 25mM sodium phosphate at pH 7.0, and mobile phase B: 25% isopropanol, 18.75mM sodium phosphate at pH 7.0. PLRP analysis used mobile phase A: 0.1% trifluoroacetic acid in water, and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Prior to PLRP analysis, samples were denatured by the addition of 50mM DTT, 4M guanidine HCl (final concentrations), and heating at 37°C for 30 minutes. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC, Tosoh #08541) with a mobile phase of 25 mM sodium phosphate at pH 6.8 containing 300 mM NaCl, 5% isopropanol.

[0419] Maleimide conjugation of untagged (wild-type) antibodies Antibody (5 mg / mL) was reduced with 2.5 molar equivalents of TCEP in PBS (pH 8.0), 1 mM DTPA for 90 min at 37° C. TCEP was removed and the protein was exchanged into PBS (pH 7.4), 1 mM DTPA using tangential flow filtration. Reduced antibody (3 mg / mL) was conjugated with 10 molar equivalents of maleimide-valcit-MMAE for 60 min on ice. Free drug was removed and the final ADC was exchanged into PBS (pH 7.4) using tangential flow filtration. Example 3: Toxicity Studies [Table 1]

[0420] Single-dose non-GLP rat toxicity study Male Sprague-Dawley rats (8-9 weeks old at the start of the study, 5 animals / group) were administered either vehicle alone or a non-cross-reactive polatuzumab (anti-CD79b) conjugate intravenously. ADCs were administered at either 20 mg / kg (vedotin conjugates with a DAR of approximately 4) or 40 mg / kg (all other conjugates with a DAR of approximately 2) to achieve equal payload dosing levels across groups. Dosing occurred on day 1, followed by an 11-day observation period. Blood was collected from all animals for clinical pathology on days 5 and 12, and for toxicokinetic analysis 8 hours post-dose, as well as on days 4, 7, and 12. Clinical observations were performed daily.

[0421] Single-dose non-GLP rat toxicity results: Polatuzumab ADCs bearing MMAE conjugated through five different linker types were compared for tolerability at equal payload dose levels in a rat study. Conjugates with the Compound 5 linker payload were superior to all other ADCs (most similar to vehicle control treated animals) with respect to effects on hematopoietic cell populations and liver function tests (AST and ALT).

[0422] FIG. 2 shows a graph of lymphocyte populations in rats 5 days after administration.

[0423] FIG. 3 shows a graph of circulating aspartate aminotransferase (AST) levels in rats 5 days after dosing.

[0424] FIG. 4 shows a graph of circulating alanine aminotransferase (ALT) levels in rats 5 days after dosing.

[0425] FIG. 5 shows a graph of red blood cell counts in rats 5 days after administration.

[0426] FIG. 6 shows a graph of hemoglobin levels in rats 5 days after administration.

[0427] FIG. 7 shows a graph of hematocrit levels in rats 5 days after dosing. [Table 2]

[0428] FIG. 8 shows a graph from the first Granta xenograft study with a single dose of ADC on day 7.

[0429] Results from the first Granta xenograft study: Polatuzumab ADCs bearing MMAE conjugated through five different linker types, including vedotin and CT aldehyde-tagged HIPS conjugates, were compared for efficacy at equal payload dose levels in the Granta 519 xenograft study. All conjugates showed excellent efficacy after a single dose, with vedotin and compound 5 conjugates showing the longest tumor growth inhibition. [Table 3]

[0430] Figure 9 shows a graph of the second Granta xenograft study with a single 2 mg / kg dose of the ADC on day 0. The use of internal tags 58Q and 91N provided superior efficacy with half the DAR compared to the vedotin conjugate.

[0431] Results from the second Granta xenograft study: Polatuzumab ADCs conjugated to vedotin or to compound 5 at various tag sites, including CT, 58Q, and 91N, were compared for efficacy at equal antibody dose levels in the Granta519 xenograft study. All conjugates showed excellent efficacy after a single 2 mg / kg dose, with compound 5 internally tagged conjugates 58Q and 91N showing the longest tumor growth inhibition. These two conjugates showed superior efficacy compared to the vedotin conjugate despite retaining only 50% of the cytotoxic payload dose (e.g., vedotin DAR of 3.45 compared to 91N ADC DAR of 1.66). In contrast to the two selected internal tags, the efficacy of the CT tagged ADC was much weaker at equal antibody doses compared to the vedotin conjugate. This difference highlights the importance of combining a specific tag site with a specific linker payload to achieve the best results. [Table 4]

[0432] Multiple Dose Non-GLP Rat Toxicity Study #1 Male Sprague-Dawley rats (8-9 weeks old at the start of the study, 5 animals / group) were administered intravenously with vehicle alone or either non-cross-reactive polatuzumab (anti-CD79b) vedotin or aldehyde-tagged HIPS conjugates, each with a DAR of approximately 4. Dosing was weekly for a total of four doses (days 1, 8, 15, and 22). Animals were observed for 7 days after the last dose. Body weights were recorded four times per week. Blood was collected for clinical pathology 4 days after dosing (for all doses). Clinical observations were performed daily.

[0433] Multi-Dose Non-GLP Rat Toxicity Study #1 Results: Polatuzumab ADCs conjugated to either vedotin or Compound 5 at the CH1 / CT tag site were compared for tolerability at equal payload / equal antibody dose levels in a multi-dose rat study. While rats receiving Compound 5 ADC showed similar results to the vehicle control group over a period of several weeks, rats receiving the vedotin conjugate showed significant myelosuppression with reductions in white and red blood cell parameters evident after the first dose and worsening over time.

[0434] FIG. 10 shows a graph of circulating neutrophil counts in rats administered vehicle or ADC repeatedly.

[0435] FIG. 11 shows a graph of circulating monocyte counts in rats repeatedly administered vehicle or ADC.

[0436] FIG. 12 shows a graph of red blood cell counts in rats administered vehicle or ADC repeatedly.

[0437] FIG. 13 shows a graph of hemoglobin levels in rats repeatedly administered vehicle or ADC.

[0438] FIG. 14 shows a graph of hematocrit levels in rats repeatedly administered vehicle or ADC. [Table 5]

[0439] Multiple Dose Non-GLP Rat Toxicity Study #2 Male Sprague-Dawley rats (8-9 weeks old at the start of the study, 5 rats / group) were administered intravenously either vehicle alone or a Nectin-4 conjugate made using an antibody carrying the variable region of the rat cross-reactive antibody Enfortumab. The ADCs tested were Nectin-4 Vedotin and Nectin-4 CH1 / CT Compound 5. Dosing at 10 mg / kg was weekly for a total of four doses (days 1, 8, 15, and 22). Animals were observed for 7 days after the last dose. Body weights were recorded four times a week. Blood was collected from all animals for clinical pathology on days 5, 12, 19, and 26 (for all doses), and toxicokinetic analysis 8 hours after dosing, and on days 4 and 7. Clinical observations were performed daily. The scale of the Clinical Observation Scoring System, ranging from 0 (normal) to 3 (severe), is shown in Table 1. [Table 6] TIFF2024529466000077.tif79162

[0440] Multiple Dose Non-GLP Rat Toxicity Study #2 Results: Enfortumab ADCs conjugated to either vedotin or Compound 5 at the CH1 / CT tag site were compared for tolerability at equal payload / equal antibody dose levels in a multiple dose rat study. One of the most striking observations from this study was the large number of clinical observations noted in the vedotin-treated group. Most of the observations were related to skin lesions. In contrast, no clinical observations were noted in the Compound 5-treated group (Figure 15). The lack of clinical observations within the Compound 5 treatment group was unexpected, considering that both vedotin and Compound 5 ADCs release the same payload (free MMAE). Previously, it was believed that the improved tolerability conferred by the Compound 5 linker was primarily related to improved stability in the circulation leading to lower off-target toxicity. However, the results of this study suggested that the Compound 5 linker may also confer additional tolerability improvements when used with ADCs with target antigen expression in healthy tissues such as the skin. This finding was novel, unexpected, and of potential therapeutic utility.

[0441] Figure 15 shows a graph of clinical observations in rats repeatedly administered rat cross-reactive Nectin-4 ADC. The arrows indicate the days of administration. There were no observations in animals administered Compound 5 conjugate, while clinical observations in the Vedotin-treated group averaged 2.5 on day 17, culminating in the death of the animals.

[0442] Example 4: Efficacy Study method NCI-H1781 xenografts with Nectin-4ADC Female BALB / c nude mice (5 mice / group) were used in the study. Animals were inoculated subcutaneously in the flank with 20 million cells in 50% PBS / 50% Matrigel. Tumors were 220 mm 3 When tumors reached an average volume of 2000 mm, animals were treated with a single intravenous dose of vehicle alone, or ADC at either 2.5 or 7.5 mg / kg. Animals were monitored twice weekly for body weight and tumor size. 3 Animals were euthanized when the dose-response curve was reached. Dose-responsive efficacy was observed for the ADC in this study.

[0443] L-82 xenotransplantation with CD30 ADC Female NOD / SCID mice (8 mice / group) were used in the study. Animals were inoculated subcutaneously in the flank with 20 million cells in 50% PBS / 50% Matrigel. Tumors grew to an average volume of 100 mm. 3 All animals were treated with a single 10 mg / kg intravenous dose of human IgG (day 0) when tumors reached 2000 mm. Animals were then treated with vehicle alone, unconjugated antibody (3 mg / kg), or ADC at 1.5 or 3 mg / kg on day 1. Animals were monitored twice weekly for body weight and tumor size. 3 The animals were euthanized when the ADC reached a high serum concentration.

[0444] Figure 17 shows a graph of the L-82 xenograft study with the listed anti-CD30 ADCs at a single intravenous dose on day 0. VH4 / VL4 Compound 8 (RED-601) uses an internal 91N tag and delivers half the payload dose compared to Adcetris. At 50% ADC dose (1.5 mg / kg) and equivalent dose (3 mg / kg), VH4 / VL4 Compound 8 was equally effective compared to Adcetris, with all treatment groups showing 8 complete responses out of 8 mice / group. VH4 / VL4 antibody alone had minimal activity.

[0445] Figure 18 shows a graph of a Karpas299 xenograft study with a single intravenous dose of the listed anti-CD30 ADCs on day 0. VH4 / VL4 Compound 8 (RED-601) uses an internal 91N tag and delivers half the payload dose compared to Adcetris. At 50% ADC dose (1.5 mg / kg) and equivalent dose (3 mg / kg), VH4 / VL4 Compound 8 produced 5 / 6 and 6 / 6 complete responses compared to Adcetris, and Adcetris produced 6 / 6 complete responses, albeit at twice the payload amount compared to VH4 / VL4 Compound 8. VH4 / VL4 antibody alone had minimal activity.

[0446] Figure 19 shows a graph of the NCI-H1781 xenograft study with the listed anti-Nectin-4 ADCs at a single 2.5 or 7.5 mg / kg intravenous dose on day 0. Both VH4 / VL1 Compound 8 (RED-601) and VH4 / VL5 Compound 8 use an internal 91N tag and deliver half the payload dose compared to Padcev. The isotype control ADC had minimal activity.

[0447] Example 5: Toxicokinetic sample analysis method Total antibody and total ADC concentrations were quantified by ELISA as previously described and diagrammed in FIG. 20. For total antibodies, conjugates were captured with anti-human IgG specific antibody and detected with anti-human Fc specific antibody conjugated with HRP. For total ADCs, conjugates were captured with anti-human Fab specific antibody and detected with mouse anti-maytansine primary antibody followed by anti-mouse IgG subclass 1 specific secondary antibody conjugated with HRP. Bound seco...

Claims

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

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

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

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

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

6. T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ is —CO—, T ３ However, (C １ ~C １２ ) alkyl, and V ３ is —CO—, and d, e, and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ But it doesn't exist, T ３ However, (PEG) ｎ and V ３ is —CO—, and d, e, and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ But it doesn't exist, T ３ However, (PEG) ｎ and V ３ is —CO—, T ４ However, (AA) ｐ and V ４ But it doesn't exist, T ５ But PABC and V ５ does not exist, and f is 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is -CONH-, T ２ However, (PEG) ｎ and V ２ is —CO—, T ３ However, (AA) ｐ and V ３ But it doesn't exist, T ４ But PABC and V ４ does not exist, and e and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is an amino acid analogue, and V ２ is —NH—, T ３ However, (PEG) ｎ and V ３ is —CO—, T ４ However, (AA) ｐ and V ４ But it doesn't exist, T ５ But PABC and V ５ does not exist, and The conjugate of claim 2 , wherein f is 0.

7. the linker L has a structure selected from: 【Chemistry 5】 【change】 During the ceremony, 【Chemistry 6】 represents the bond of L to N in formula (I), * represents W １ 2. The conjugate of claim 1, wherein L represents the bond to

8. 2. The conjugate of claim 1, wherein the drug is monomethyl auristatin E (MMAE).

9. The conjugate may comprise: 【Chemistry 7】 【change】 2. The conjugate of claim 1, selected from the group consisting of:

10. The conjugate of claim 1 , wherein the antibody is an IgG1 antibody.

11. The conjugate of claim 10, wherein the antibody is an IgG1 kappa antibody.

12. 2. The conjugate of claim 1, wherein the antibody comprises a sequence having fGly', where fGly' is an amino acid residue coupled to the drug through the linker.

13. The conjugate of claim 1 , wherein the sequence is located at the C-terminus of the heavy chain constant region of the antibody.

14. The conjugate of claim 1 , wherein the sequence is located within the light chain constant region of the antibody.

15. The conjugate of claim 1 , wherein the sequence is located within the heavy chain CH1 region of the antibody.

16. The conjugate of claim 1 , wherein the sequence is located within the heavy chain CH2 region of the antibody.

17. The conjugate of claim 1 , wherein the sequence is located within the heavy chain CH3 region of the antibody.

18. A compound of formula (III): 【Chemistry 8】 During the ceremony, Z is CR ４ or N, R ２ and R ３ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R ２ and R ３ are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R ４ are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L is a linker, W １ But it is a drug, a compound.

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

20. T １ However, (C １ ~C １２ ) alkyl and substituted (C １ ~C １２ ) alkyl; T ２ , T ３ , T ４ , T ５ , and T ６ each independently represents a covalent bond, (C １ ~C １２ ) alkyl, substituted (C １ ~C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ OH) ｍ -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines, and esters; V １ , V ２ , V ３ , V ４ , V ５ , and V ６ each independently represents a covalent bond, —CO—, or —NR １５ -, -NR １５ (CH ２ ) ｑ -, -NR １５ (C ６ H ４ ) -, -CONR １５ -, -NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ NR １５ -, -NR １５ SO ２ -, and -P(O)OH-; (PEG) ｎ but, 【Chemistry 9】 wherein n is an integer from 1 to 30; EDA has the following structure: 【Chemistry 10】 wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) 【Chemistry 11】 and Each R １２ are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R １２ 20. The compound of claim 19, wherein the groups can be cyclically linked to form a piperazinyl ring.

21. 20. The compound of claim 19, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

22. 22. The compound of claim 21, wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

23. T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ is —CO—, T ３ However, (C １ ~C １２ ) alkyl, and V ３ is —CO—, and d, e, and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ But it doesn't exist, T ３ However, (PEG) ｎ and V ３ is —CO—, and d, e, and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is 4AP, and V ２ But it doesn't exist, T ３ However, (PEG) ｎ and V ３ is —CO—, T ４ is AA and V ４ But it doesn't exist, T ５ But PABC and V ５ does not exist, and f is 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is -CONH-, T ２ However, (PEG) ｎ and V ２ is —CO—, T ３ is AA and V ３ But it doesn't exist, T ４ But PABC and V ４ does not exist, and e and f are each 0, or T １ However, (C １ ~C １２ ) alkyl, and V １ is —CO—, T ２ is an amino acid analogue, and V ２ is —NH—, T ３ However, (PEG) ｎ and V ３ is —CO—, T ４ is AA and V ４ But it doesn't exist, T ５ But PABC and V ５ does not exist, and 20. The compound of claim 19, wherein f is 0.

24. the linker L has a structure selected from: 【Chemistry 12】 During the ceremony, 【Chemistry 13】 represents the bond of L to N in formula (I), * represents W １ 19. The compound of claim 18, wherein L represents the bond to:

25. 19. The compound of claim 18, wherein the drug is MMAE.

26. The compound is 【Chemistry 14】 19. The compound of claim 18 selected from the group consisting of:

27. 1. A pharmaceutical composition comprising: A conjugate according to claim 1; and a pharmaceutically acceptable excipient.

28. 28. The pharmaceutical composition of claim 27 for use in a method of treating cancer in a subject.

29. 29. The composition of claim 28, wherein the cancer is breast cancer, ovarian cancer, lung cancer, or gastric cancer.

30. 28. The pharmaceutical composition of claim 27 for use in a method of delivering a drug to a target site in a subject.

Images