Drug linkers and antibody conjugates thereof

Abstract

Disclosed herein are antibody-drug conjugates (ADCs), drug-linkers for ADCs, and tunable phosphate-based linkers. The ADCs of the present disclosure include antibodies, such as anti-CD70 antibodies, conjugated to duocarmycin analogs via phosphate-based uniters. Also disclosed are methods and compositions for using the ADCs in inhibiting, preventing, or treating diseases or conditions, such as cancer.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 378,852, filed October 7, 2022, the entire contents of which are hereby incorporated herein in their entirety.

[0002] (Sequence Listing) This application has been submitted in XML format and contains a Sequence Listing, which is hereby incorporated by reference in its entirety. The XML copy created on October 6, 2023 is designated AMBX-024500PCT and is 78,546 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to antibody-drug conjugates (ADCs), cytotoxic duocarmycin analog drugs, and drug-linkers. Specifically, the present invention relates to unnatural amino acid-containing antibodies conjugated to duocarmycin analogs and drug-linkers containing PEGylated phosphate-based linkers. The present invention also relates to methods of using the ADCs, drugs, and drug-linkers, including the treatment of cancer. [Background technology]

[0004] Antibody-drug conjugates (ADCs) are a powerful class of therapeutic constructs that advance the field of cancer therapy by enabling the targeted delivery of cytotoxic drugs to target cells, such as cancer cells. Currently, only a few ADCs have been approved for therapeutic use, including gemtuzumab ozogamicin for AML (since withdrawn from the market), brentuximab vedotin for ALCL and Hodgkin lymphoma, and trastuzumab emtansine for HER2-positive metastatic breast cancer (Verma et al., N Engl J Med 367:1783-91, 2012; Bross et al., Clin Cancer Res 7:1490-96, 2001; Francisco et al., Blood 102:1458-65, 2003), and sacituzumab govitecan for metastatic triple-negative breast cancer (TNBC) (Zaman et al., OncoTargets and Therapy 12:1781-1790, 2019). However, ADCs face challenges due to their lack of therapeutic index and toxicity. Linker technology for conjugating cytotoxic drugs to antibodies affects the stability of ADCs in the systemic circulation. Therefore, there is a need in the art for designing improved linkers, such as phosphate-based linkers, and drug design for antibody conjugation.

[0005] The present disclosure provides phosphate-based linkers with tunable stability for intracellular delivery of drug payloads. The phosphate-based linkers have differentially tunable stability in the blood relative to the intracellular environment and can further include self-immolative linkers. Antibody-drug conjugates containing these linkers are stable in the circulation (plasma / blood) but reactive or cleavable in intracellular compartments such as the lysosomal compartment, making them useful for intracellular delivery, the rate of which depends on the structure of the regulating element.

[0006] Cluster of differentiation 70 (CD70) is a member of the tumor necrosis factor superfamily and a ligand for CD27 (Goodwin, R.G. et al., Cell, 73:447-456 (1993); Hintzen, R.Q. et al., Int Immunol, 6:477-480 (1994)). CD70 was first identified on activated T and B lymphocytes. Binding of CD70 to CD27 on activated lymphocytes signals costimulation of T cells, B cells, and natural killer (NK) cells (Grewal, I. S., Expert Opin Ther Targets, 12(3):341-351 (2008); Borst, J. et al., Curr Opin Immunol., 17(3):275-281 (2005)) and regulates cell differentiation and T helper 1 / 2 switching (Wajant, H., Expert Opin Ther Targets, 20(8):959-973 (2016)). The primary amino acid sequence of CD70 predicts it to be a transmembrane type II protein, with its carboxyl terminus exposed extracellularly and its amino terminus found on the cytoplasmic side of the plasma membrane. Human CD70 consists of a 20-amino acid cytoplasmic domain, an 18-amino acid transmembrane domain, and a 155-amino acid extracytoplasmic domain with two potential N-linked glycosylation sites (Bowman et al., J Immunol, 152:1756-1761 (1994); Goodwin et al., Cell, 73:447-456 (1993)).

[0007] CD70 expression has been reported in different types of cancer, including lymphomas, carcinomas, and tumors of neural origin. Among malignant B cells, 71% of diffuse large B-cell lymphomas, 33% of follicle center lymphomas, 25% of mantle lymphomas, and 50% of B-CLLs have been reported to express CD70 (Lens et al., 1999, Br J Haematol, 106:491-503). CD70 has also been detected in brain tumor cells, particularly glioma cell lines, solid human gliomas, and meningiomas (Held-Feindt and Mentlein, Int J Cancer, 98:352-56 (2002); Wischlusen et al., Can Res, 62:2592-2599 (2002)). CD70 is frequently expressed in renal cell carcinoma (RCC, 87%) and non-Hodgkin's lymphoma (NHL, 77%) (Tannir, NM et al., Invest New Drugs, 32(6):1246-1257(2014)), but is minimally expressed in normal tissues (Nakae, R. et al., Am J Obstet Gynecol., 224(2):197(2021)).

[0008] Anti-CD70 antibodies and antibody-drug conjugates (ADCs), and methods for making and using them to treat diseases such as cancer, are disclosed in WO 2013 / 192360 A1, the entire contents of which are incorporated herein by reference in their entirety.

[0009] Several clinical trials evaluating anti-CD70 agents (e.g., antibodies with enhanced antibody-dependent cell-mediated cytotoxicity, ADCs, and chimeric antigen receptor (CAR) T-cell therapy) are being investigated in malignancies that exhibit high CD70 expression. Previous studies have shown that anti-CD70 monoclonal antibodies (mAbs) and anti-CD70 ADCs exhibit anti-tumor effects in xenograft models of CD70-positive malignancies, such as lymphoma, NHL, and RCC (Israel, BF et al., Mol Cancer Ther., 4(12), 2037-2044 (2005); Law, CL et al., Cancer Res., 66:2328-2337 (2006); McEarchern, JA et al., Blood, 109(3):1185-92 (2007)). Based on the results of preclinical studies, two separate phase 1 trials of SGN-75 (an anti-CD70 mAb conjugated to maleimidocaproyl-monomethyl auristatin F (MMAF)) were conducted in patients with CD70-positive relapsed / refractory NHL or metastatic RCC; however, SGN-75 showed modest efficacy against these diseases and had several intolerable side effects (Tannir, NM et al., Invest New Drugs, 32(6):1246-1257 (2014)). An additional anti-CD70 ADC, SGN-CD70A (an anti-CD70 mAb conjugated to a pyrrolobenzodiazepine dimer), was introduced into a Phase 1 clinical trial (Pal, SK et al., Cancer, 125(7):1124-1132(2019)), but the SGN-CD70A Phase 1 trial was discontinued in 2018.

[0010] Duocarmycin SA is a highly potent cytotoxic natural product that can bind to the DNA minor groove and induce sequence-selective alkylation of double-stranded DNA. Duocarmycin-based ADCs include BMS-936561 (MDX-1203), which contains an anti-CD70 antibody conjugated to the duocarmycin derivative MED-A via a maleimide-containing citrulline-valine dipeptide linker (Wang H. et al. (2016) Biopharm Drug Disp 37(2):93-106, Owonikoko TK et al., Cancer Chemother Pharmacol (2016) 77(1):155-162). The cessation of development of BMS-936561 / MDX-1203 illustrates the challenges facing duocarmycin-based ADCs (Hang-Ping Y. et al., Drug Discov Today (2021) 26(8):1857-1874).

[0011] There remains a need for anti-CD70 ADCs that are constructed in a manner that allows them to exert clinically useful cytotoxic, cytostatic, or immunosuppressive effects on CD70-expressing cells, particularly without undesirable effects on non-CD70-expressing cells. Such ADCs would be useful therapeutic agents for CD70-expressing cancers or immune disorders mediated by CD70-expressing cells. The present invention provides such ADCs for use in immunology and oncology. Summary of the Invention

[0012] The present invention provides novel drugs and drug-linkers suitable for antibody conjugation, wherein the drugs are duocarmycin analogs and the drug-linkers comprise phosphate-based linkers. The present invention further provides ADCs comprising the phosphate-based drug-linkers. The drugs, drug-linkers, and ADCs are suitable for treating diseases and conditions in human subjects in need thereof, including cancer.

[0013] In some general embodiments, the compound of formula (I):

[0014] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; A has the following structures, formulas (a), (b), (c), and (d):

[0015] [ka] is a bicyclic ring system selected from the group consisting of During the ceremony, each X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 3 is C, each X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(Rb ), -C(O)SR c or -S(O) m (R s ) and each X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m(R s ) and each X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 8 is C, each X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R sare independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 or a salt thereof.

[0016] In some embodiments, AH (the corresponding amine of moiety A) has a ClogP value of at least about 1.

[0017] In some embodiments, A has the structure of formula (a) and the compound has the structure of formula (Ia):

[0018] [ka] or a salt thereof.

[0019] In some embodiments, A has the structure of formula (b) and the compound has the structure of formula (Ib):

[0020] [ka] or a salt thereof.

[0021] In some embodiments, A has the structure of formula (c) and the compound has the structure of formula (Ic):

[0022] [ka] or a salt thereof.

[0023] In some embodiments, A has the structure of formula (d) and the compound has the structure of formula (Id):

[0024] [ka] or a salt thereof.

[0025] In some embodiments, a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d) is provided, wherein: R is H or LW, where L is a linker and W is a reactive moiety; X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 5 is C(R5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 7 is C(R 7 ) or N, and R 7is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each m is independently 0, 1, 2, or 3.

[0026] In some further aspects, compounds of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d) are provided, wherein: X1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 8 is C, X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0027] In some embodiments, a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d) is provided, wherein: X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b is H, X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b is H, X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, X 8 is C, X 9 is CH2, if present.

[0028] In some embodiments, each said heteroalkyl is alkoxy.

[0029] In some embodiments, a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d) is provided, wherein: X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b is H, X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b is H, X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, X 5 is C(R 5 ) or N, and R 5 is H, halogen or alkoxy, X 6 is C(R 6 ) or N, and R 6 is H, halogen or alkoxy, X 7 is C(R 7 ) or N, and R 7 is H, X 8 is C, X 9 is CH2, if present.

[0030] In some embodiments, X 5 is C(R 5 ) or N, and R 5 is H or alkoxy, and X 6 is C(R 6) or N, and R 6 is H or alkoxy.

[0031] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N.

[0032] In some embodiments, X 4 and X 7 At least one of X is CH. 4 and X 7 Each of the is CH.

[0033] In some embodiments, R 5 and R 6 In some further embodiments, at least one of the alkoxy groups is independently selected from the group consisting of -OR k and each R k are independently heterocyclyl or -N(R d )(R e ), wherein the heterocyclyl contains at least one nitrogen atom and each R d and R e is independently H, alkyl, alkenyl, or alkynyl. In some embodiments, each alkoxy is selected from the group consisting of -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -OCH2CH2N(CH3)2,

[0034] [ka] is selected from the group consisting of:

[0035] In some embodiments, the compounds of the disclosure are compounds of formula (I), where R is H. In some embodiments, the compounds of the disclosure are compounds of formula (Ia), where R is H. In some embodiments, the compounds are compounds of formula (Ib), where R is H. In some embodiments, the compounds are compounds of formula (Ic), where R is H. In some embodiments, the compounds are compounds of formula (Id), where R is H.

[0036] In some embodiments, the compound is of formula (Ia), wherein R is H, and the compound is

[0037] [ka] and salts thereof.

[0038] In some embodiments, the compound is of formula (I), where R is LW. In some embodiments, the compound of the present disclosure is of formula (Ia), where R is LW. In some embodiments, the compound is of formula (Ib), where R is LW. In some embodiments, the compound is of formula (Ic), where R is LW. In some embodiments, the compound is of formula (Id), where R is LW.

[0039] In certain embodiments, L is a phosphate-based linker. In some embodiments, the phosphate-based linker has the following structure:

[0040] [ka] and a phosphate-based moiety having the formula: * indicates a connection to the —O— atom at position R of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), and L further comprises at least one additional moiety, and the wavy line in the phosphate-based moiety indicates a connection to one of the at least one additional moiety, and the at least one additional moiety is unsubstituted alkylene, substituted alkylene, -(alkylene-O)-, optionally substituted arylene, -O-, -C(O)-, -N(R w )-, -S(O) 0-2 -, a water-soluble polymer, and an amino acid; w is independently H or C1-C8 alkyl, and combinations thereof. In some embodiments, each at least one additional moiety is independently unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R w )-, a water-soluble polymer, and an amino acid; w is independently H or C1-C8 alkyl, and combinations thereof.

[0041] In some embodiments, L is selected from the group of linkers in Table 6. In some other embodiments, L is selected from the group of linkers in Table 7. In some other embodiments, L is selected from the group of linkers in Table 8. In some embodiments, L has the following structure:

[0042] [ka] wherein * indicates a connection to the -O- atom at position R of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), and + indicates a connection to W. In some other embodiments, L is one of the following structures:

[0043] [ka] wherein * indicates a connection to the -O- atom at position R of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), and + indicates a connection to W. In some other embodiments, L is one of the following structures:

[0044] [ka] wherein T is a water-soluble polymer and R t is H or methyl, * indicates a connection to the -O- atom at position R of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), and + indicates a connection to W. In some embodiments, the water-soluble polymer is a (polyethylene) glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight within the range of about 100 Da to about 100,000 Da, about 100 Da to about 10,000 Da, about 100 Da to about 5,000 Da, or about 100 Da to about 1,000 Da. In some embodiments, the PEG moiety is -(CH2CHO) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) nCH3, and n is 8, 9, 10, 11, or 12.

[0045] In some embodiments, the reactive moiety W is —N, —OH, —SH, —NH(R j ), -C(O)R q , -C(O)OR x , -C(O)CH2NH2, activated ester, -O-NH2, maleimide, tetrazine, alkyne, cyclooctyne or (E)-cyclooctene, R j is H or unsubstituted alkyl, and R q is unsubstituted alkyl, and R x is H, an unsubstituted alkyl, or a carboxylic acid protecting group. In some further embodiments, the reactive moiety W is

[0046] [ka] -N3, -OH, -SH, -NH(R j ), -C(O)R q , -C(O)OR x , an activated ester, —O—NH, and an optionally substituted monocyclic or polycyclic group including cyclooctyne, wherein R j is H or unsubstituted C1-C6 alkyl, and R q is unsubstituted C1-C6 alkyl, and R x is H, unsubstituted C1-C6 alkyl or a carboxylic acid protecting group, and R f is H or unsubstituted C1-C6 alkyl; s is 0, 1, 2, 3, 4, 5, or 6; and t is 0, 1, 2, 3, 4, 5, or 6.

[0047] In some embodiments, W is -ONH2.

[0048] In some embodiments, a compound of Formula (Ia) is provided, wherein R is LW, and the compound is

[0049] [ka]

[0050] [ka] and salts thereof.

[0051] In some other general aspects, the present disclosure provides a compound of formula (II):

[0052] [ka] (In the formula, Ab is an antibody, the Ab comprising one or more unnatural amino acids; L is a linker, E is the moiety linking Ab and L, d is an integer from 1 to 10, A has the following structures, formulas (a), (b), (c), and (d):

[0053] [ka] is selected from the group consisting of During the ceremony, each X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 3 is C, each X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(Rb ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 8 is C, each X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, AH (the corresponding amine of moiety A) has a ClogP value of at least about 1.

[0055] In some embodiments, an ADC of formula (II) is provided, wherein A is formula (a). In some embodiments, an ADC of formula (II) is provided, wherein A is formula (b). In some embodiments, an ADC of formula (II) is provided, wherein A is formula (c). In some embodiments, an ADC of formula (II) is provided, wherein A is formula (d).

[0056] In some embodiments, an ADC of formula (II) is provided, wherein: X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a)(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 8 is C, X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0057] In some embodiments, an ADC of formula (II) is provided, wherein: X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b is H, X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b is H, X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, X 8 is C, X 9 is CH2, if present.

[0058] In some embodiments, each said heteroalkyl is alkoxy. Thus, in some embodiments, an ADC of Formula (II) is provided, wherein: X 1 is C(R1a )(R 1b ) and each R 1a and R 1b is H, X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b is H, X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, X 5 is C(R 5 ) or N, and R 5 is H, halogen or alkoxy, X 6 is C(R 6 ) or N, and R 6 is H, halogen or alkoxy, X 7 is C(R 7 ) or N, and R 7 is H, X 8 is C, X 9 is CH2, if present.

[0059] In some embodiments, X 5 is C(R 5 ) or N, and R 5 is H or alkoxy, and X 6 is C(R 6 ) or N, and R 6 is H or alkoxy.

[0060] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some other embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N.

[0061] In some embodiments, X 4 and X 7 At least one of X is CH. 4 and X 7 Each of the is CH.

[0062] In some embodiments, R 5 and R 6 At least one of is alkoxy.

[0063] In some embodiments, d is 1, 2, 3, or 4. In some embodiments, d is 2. In some embodiments, d is 4.

[0064] In some embodiments, ADCs of Formula (II) are provided, wherein L is a phosphate-based linker. In some embodiments, the phosphate-based linker has the following structure:

[0065] [ka] and a phosphate-based moiety having the formula: * indicates a connection to the -O- atom at position L of formula (II), where L further comprises at least one additional moiety, and the wavy line in the phosphate-based moiety indicates a connection to one of the at least one additional moiety. In some embodiments, each at least one additional moiety is independently selected from unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R w )-, a water-soluble polymer, and an amino acid; w is independently H or C1-C8 alkyl, and combinations thereof. In some embodiments, L is selected from the group of linkers in Table 6. In some other embodiments, L is selected from the group of linkers in Table 7. In some other embodiments, L is selected from the group of linkers in Table 8. In some embodiments, L has the following structure:

[0066] [ka] wherein * indicates a connection to the -O- atom at position L of formula (II), and + indicates a connection to E. In some other embodiments, L has the following structure:

[0067] [ka] wherein * indicates a connection to the -O- atom at position L of formula (II), and + indicates a connection to E. In some other embodiments, L has the following structure:

[0068] [ka] wherein T is a water-soluble polymer and R t is H or methyl, * indicates a connection to the -O- atom at position L in formula (II), and + indicates a connection to E. In some embodiments, the water-soluble polymer is a (polyethylene) glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight within the range of about 100 Da to about 100,000 Da, about 100 Da to about 10,000 Da, about 100 Da to about 5,000 Da, or about 100 Da to about 1,000 Da. In some embodiments, the PEG moiety is -(CH2CHO) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, where n is 8, 9, 10, 11, or 12. In some embodiments, n is 8. In some embodiments, n is 12.

[0069] In some embodiments, E comprises an amide, an ester, a thioester, a pyrrolidine-2,5-dione, an oxime, a 1,2,3-triazole, or a 1,4-dihydropyridazine, wherein the 1,2,3-triazole and the 1,4-dihydropyridazine are each optionally fused to an 8-membered ring.

[0070] [ka] is selected from the group consisting of In the formula, each R j are independently H or unsubstituted C1-C6 alkyl, and each R q is independently an unsubstituted C1-C6 alkyl, and each R f is independently H or unsubstituted C1-C6 alkyl, each s is independently 0, 1, 2, 3, 4, 5, or 6, each t is independently 0, 1, 2, 3, 4, 5, or 6, each + indicates a connection to L, and each wavy line indicates a connection to Ab. In some embodiments, E is

[0071] [ka] and In the formula, R q is unsubstituted C1-C6 alkyl. In some embodiments, R q is methyl.

[0072] In some embodiments, E links L to the unnatural amino acid of the Ab.

[0073] In some embodiments, R q is the Ab-encoded unnatural amino acid methyl. In some embodiments, the unnatural amino acid is para-acetyl-L-phenylalanine (pAF) and R q is the methyl group of the pAF acyl group.

[0074] In some embodiments, the Ab is configured to bind to an antigen, such as PD-1, PD-L1, PSMA, CD70, CD3, HER2, HER3, TROP2, GPC3, VEGFR, EGFR, c-Met (HGFR), CD19, CD22, CD25 (IL-2R alpha), CD30, CD33, CD37, CD46, CD48, CD56 (NCAM-1), CD71 (Transferrin R), CD74, CD79b, CD123 (IL-3R alpha), CD138 (Syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP3), CD205 (LY75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, EpCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY2c, NaPi2b, Flt-1, Flt-3, folate receptor alpha, tissue factor (TF), CA6, MUC1, MUC16 (CA-125), BCMA, SLAMF7 (CS1), TIM1, CanAg, Ckit (CD117), Eph A2, Nectin4, SLTRK6, FGFR2, LYPD3 (C4.4a), Cadherin 3, 5T4 (TPBG), STEAP1, PTK7, Ephrin-A4, LIV-1 (SLC39A6 or ZIP6), SLC1A5, TENB2, ETBR, Integrin v3, Cripto, AGS-5 (SLC44A4), LY6E, AXL, LAMP1, LRRC15, TNF-alpha, and MN / CA IX. In some embodiments, the antigen is TROP2, CD70, HER2, PSMA, HER3, or GPC3.

[0075] In some aspects, the Ab is an anti-CD70 antibody comprising a sequence listed in Table 2. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 25. In some other embodiments, the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD70 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 19. In some aspects, the anti-CD70 antibody comprises two heavy chains, each having the amino acid sequence of SEQ ID NO: 20, and two light chains, each having the amino acid sequence of SEQ ID NO: 19. In some other aspects, the anti-CD70 antibody comprises two heavy chains, each having the amino acid sequence of SEQ ID NO: 25, and two light chains, each having the amino acid sequence of SEQ ID NO: 19.

[0076] In some other embodiments, the Ab is an anti-TROP2 antibody comprising a sequence listed in Table 1. In some embodiments, the anti-TROP2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-TROP2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 4.

[0077] In some other embodiments, the Ab is an anti-HER2 antibody comprising a sequence listed in Table 3.

[0078] In some embodiments, the anti-HER2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-HER2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 30.

[0079] In some other embodiments, the Ab is an anti-PSMA antibody comprising a sequence listed in Table 4.

[0080] In some embodiments, the anti-PSMA antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 40.

[0081] In some other embodiments, the Ab is an anti-HER3 antibody comprising a sequence listed in Table 5.

[0082] In some embodiments, the anti-HER3 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 47.

[0083] In some embodiments, the antibody (Ab) comprises two heavy chains and one unnatural amino acid is incorporated into each heavy chain.

[0084] In some embodiments, the unnatural amino acid is para-acetyl-L-phenylalanine.

[0085] In some other general aspects, the disclosure provides pharmaceutical compositions comprising a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), or an ADC of Formula (II), and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent, or excipient.

[0086] In some other general aspects, the disclosure provides methods of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), or an ADC of Formula (II), or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula I(d), or an ADC of Formula (II). In some aspects, the disease or condition is cancer. In some aspects, the cancer is a CD70-expressing cancer. In some aspects, the cancer is renal cell carcinoma. In some other aspects, the cancer is a hematological cancer. In some aspects, the hematological cancer is leukemia, lymphoma, or myeloma.

[0087] It is understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein.

[0088] Incorporation by Reference All publications, patents, patent applications, and / or other documents mentioned herein are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was specifically and individually indicated to be incorporated by reference for all purposes, and for the purposes of describing and disclosing, for example, the compositions and other methodologies described in the publications, patents, patent applications, and / or other documents, that may be used in connection with the invention(s) described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. [Brief explanation of the drawings]

[0089] [Figure 1A] 1A shows an evaluation of the in vitro cytotoxic activity of duocarmycin analog compounds and anti-CD70 ADCs against the CD70-positive cell line 786-O (FIG. 1A) and the CD70-negative cell line NCI-H929 (FIG. 1B). [Figure 1B] 1A shows an evaluation of the in vitro cytotoxic activity of duocarmycin analog compounds and anti-CD70 ADCs against the CD70-positive cell line 786-O (FIG. 1A) and the CD70-negative cell line NCI-H929 (FIG. 1B). [Figure 2A] 2A and 2B show evaluation of the in vitro cytotoxic activity of duocarmycin analog compounds and anti-GPC3 ADCs against the GPC3-positive cell line HepG2 (FIG. 2A) and the GPC3-negative cell line SUN499 (FIG. 2B). [Figure 2B]2A and 2B show evaluation of the in vitro cytotoxic activity of duocarmycin analog compounds and anti-GPC3 ADCs against the GPC3-positive cell line HepG2 (FIG. 2A) and the GPC3-negative cell line SUN499 (FIG. 2B). DETAILED DESCRIPTION OF THE INVENTION

[0090] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular methodology, compositions, or biological systems, which can, 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 in any way.

[0091] While various embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention, and it is intended to cover thereby all methods and structures within the scope of the claims and their equivalents.

[0092] definition Unless otherwise defined herein or below in the remainder of the specification, 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. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0093] 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 the invention described herein belongs. Various methods, materials, and the like similar or equivalent to those described herein can be used in the practice or testing of the invention described herein.

[0094] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the chemistry, chemical syntheses, compositions and other methodologies described in the publications, which might be used in connection with the invention(s) described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application.

[0095] chemical terms It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0096] The term "acyl," as used herein, refers to -C(O)-alkyl, as defined herein, and is exemplified by acetyl (-C(O)CH), trifluoroacetyl, propionyl, and butanoyl. Exemplary unsaturated acyl groups contain 1 to 6, 1 to 11, or 1 to 21 carbons.

[0097] The term "alkyl," as used herein, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1 to 20 carbon atoms. Non-limiting examples of alkyl groups include alkyl groups containing 1 to 16 carbon atoms (C 1~16 alkyl), 1 to 10 carbon atoms (C 1~10 alkyl), 1 to 6 carbon atoms (C 1~6 Examples of aliphatic hydrocarbon radicals include alkyl, 4 carbon atoms (e.g., n-butyl, iso-butyl, sec-butyl, t-butyl), 3 carbon atoms (e.g., isopropyl or n-propyl), 2 carbon atoms (ethyl), and 1 carbon atom (methyl). Alkylene is a divalent alkyl group.

[0098] The term "alkenyl," as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon double bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).

[0099] The term "alkoxy," as used herein, alone or in combination with other groups, refers to an alkyl group having a single bond to oxygen. Non-limiting examples of alkoxy groups of the present disclosure include methoxy (-OMe) and ethoxy (-OEt). The alkoxy groups of the present disclosure are optionally substituted. In some embodiments, the alkoxy groups of the present disclosure are optionally heterocyclyl or -N(R d )(R e ) and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0100] The term "alkynyl," as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).

[0101] The term “amino” as used herein refers to —N(R N1 )2, wherein each R N1 are independently H, OH, NO2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2 , an N-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), and these enumerated R N1 Each of the groups can be optionally substituted, or two R N1 are bonded to form an alkylene or heteroalkylene, and each RN2 is independently H, alkyl, or aryl. The amino group of the present invention can be unsubstituted amino (i.e., —NH) or substituted amino (i.e., —N(R N1 2) can be.

[0102] The term "aryl," as used herein, refers to an aromatic mono- or polycarbocyclic radical of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, 1,2-dihydronaphthyl, indanyl, and 1H-indenyl.

[0103] The term "arylalkyl," as used herein, refers to an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups are those containing 7 to 30 carbons (e.g., C), such as benzyl and phenethyl. 1~6 Alkyl C 6~10 Aryl, C 1~10 Alkyl C 6~10 Aryl, or C 1~20 Alkyl C 6~10 aryl, etc.) In some embodiments, akyl and aryl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the respective groups.

[0104] The term "azido" as used herein refers to an -N3 group.

[0105] As used herein, the term "bicyclic ring system" refers to a bicyclic moiety or molecule containing two linked rings, wherein the two rings are joined by the sharing of two or more atoms. In some embodiments, the bicyclic ring system shares two atoms. In one embodiment, the bicyclic ring system contains at least one ring nitrogen atom.

[0106] The term "cyano," as used herein, refers to a --CN group.

[0107] The term "carbocyclyl" as used herein refers to a non-aromatic C 3~12 It refers to a monocyclic, bicyclic, or tricyclic structure. The carbocyclyl structure comprises a cycloalkyl group and an unsaturated carbocyclyl radical.

[0108] The term "cycloalkyl," as used herein, refers to a saturated, non-aromatic, monovalent mono- or polycarbocyclic radical of 3 to 10, preferably 3 to 6, carbon atoms. This term is further exemplified by groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.

[0109] The term "halogen," as used herein, means a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical.

[0110] The term "heteroalkyl," as used herein, refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms has been replaced by nitrogen, oxygen, or sulfur. In some embodiments, heteroalkyl groups can be further substituted with one, two, three, or four substituents described herein for alkyl groups. Non-limiting examples of heteroalkyl groups include aminoalkyl and "alkoxy."

[0111] A heteroalkylene is a divalent heteroalkyl group.

[0112] The term "heteroalkenyl," as used herein, refers to an alkenyl group, as defined herein, in which one or more of the constituent carbon atoms has been replaced by nitrogen, oxygen, or sulfur. In some embodiments, heteroalkenyl groups can be further substituted with one, two, three, or four substituents described herein for alkenyl groups. An example of a heteroalkenyl group is "alkenoxy," which, as used herein, refers to alkenyl-O-. Heteroalkenylene is a divalent heteroalkenyl group.

[0113] The term "heteroalkynyl," as used herein, refers to an alkynyl group, as defined herein, in which one or more of the constituent carbon atoms has been replaced by nitrogen, oxygen, or sulfur. In some embodiments, heteroalkynyl groups can be further substituted with one, two, three, or four substituents described herein for alkynyl groups. An example of a heteroalkynyl group is "alkynoxy," which, as used herein, refers to alkynyl-O-. Heteroalkynylene is a divalent heteroalkynyl group.

[0114] The term "heteroaryl," as used herein, refers to an aromatic monocyclic or polycyclic radical of 5 to 12 atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or two ring carbon atoms of a heteroaryl group may be replaced by a carbonyl group. Examples of heteroaryl groups are pyridyl, pyrazolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl.

[0115] The term "heteroarylalkyl," as used herein, refers to an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups are those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 Alkyl C 2~9 Heteroaryl, C 1~10 Alkyl C 2~9Heteroaryl, or C 1~20 Alkyl C 2~9 In some embodiments, akyl and heteroaryl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the respective groups.

[0116] The term "heterocyclyl," as used herein, refers to a monocyclic or polycyclic radical having 3 to 12 atoms with at least one ring containing 1, 2, 3, or 4 ring heteroatoms selected from N, O, or S, and the ring is not aromatic. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1,3-dioxanyl.

[0117] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocyclyl group. Exemplary unsubstituted heterocyclylalkyl groups are those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 Alkyl C 2~9 Heterocyclyl, C 1~10 Alkyl C 2~9 Heterocyclyl, or C 1~20 Alkyl C 2~9 In some embodiments, the akyl and heterocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the respective groups.

[0118] The term "hydroxyl," as used herein, refers to an --OH group.

[0119] The term "carboxy" as used herein refers to a -NO2 group.

[0120] The term "thiol," as used herein, refers to a --SH group.

[0121] Alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups can be substituted or unsubstituted. If substituted, there will generally be 1 to 4 substituents, unless otherwise specified. Substituents include, for example, aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halogen (e.g., fluoro), hydroxyl, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NH or mono- or dialkylamino), azido, cyano, nitro, or thiol. Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups can also be substituted with alkyl (such as unsubstituted and substituted arylalkyl (e.g., substituted and unsubstituted benzyl)).

[0122] The compounds of the present invention may have one or more asymmetric carbon atoms and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g., racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereomeric racemates, or mixtures of diastereomeric racemates. Optically active forms can be obtained, for example, by resolution of racemates, asymmetric synthesis, or asymmetric chromatography (chromatography using a chiral adsorbent or eluent). That is, certain disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are most commonly pairs of stereoisomers whose mirror images are not superimposable because they contain asymmetrically substituted carbon atoms that act as chiral centers. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared by separating the enantiomer from a racemate using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon. Suitable techniques and / or methods for separating enantiomers of compounds described herein from racemic mixtures can be readily determined by those skilled in the art. A "racemate" or "racemic mixture" refers to a compound containing two enantiomers; such mixtures do not exhibit optical activity, i.e., they do not rotate the plane of polarized light. A "geometric isomer" refers to isomers that differ in the orientation of substituent atoms with respect to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic ring system. Atoms (other than H) on each side of a carbon-carbon double bond can be in the E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. "R", "S", "S" * "," "R *"," "E," "Z," "cis," and "trans" indicate configurations relative to the core molecule. Certain disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about a single bond, where the steric strain barrier to rotation is high enough to allow isolation of the conformers. The compounds of the invention can be prepared as individual isomers by isomer-specific synthesis or by resolution from an isomeric mixture. Classical resolution techniques include forming a salt of the free base of each isomer of the isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming a salt of the acid form of each isomer of the isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each isomer of the isomer of the isomeric pair using an optically pure acid, amine, or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either the starting materials or the final product using a variety of well-known chromatographic methods. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% optically pure by weight. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Percent optical purity is the ratio of the weights of the enantiomers, or the ratio of the weight of the enantiomer to the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer to the weight of all diastereomers. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by mole fraction relative to other stereoisomers.When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by mole fraction. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by mole fraction. Percent purity by mole fraction is the ratio of moles of enantiomers, or moles of enantiomer plus moles of its optical isomer. Similarly, percent purity by mole fraction is the ratio of moles of diastereomers, or moles of diastereomer plus moles of its optical isomer. When a disclosed compound is named or depicted by structure without indicating stereochemistry, and the compound has at least one chiral center, it should be understood that the name or structure encompasses either an enantiomer of the compound free of the corresponding optical isomer, a racemic mixture of the compound, or a mixture enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or depicted by structure without indicating stereochemistry, and the compound has two or more chiral centers, it should be understood that the name or structure encompasses a diastereomer free of the other diastereomer, multiple diastereomers free of other diastereomeric pairs, a mixture of diastereomers, a mixture of diastereomeric pairs, a mixture of diastereomers enriched in one diastereomer relative to the other, or a mixture of diastereomers enriched in one or more diastereomers relative to the other. The present invention encompasses all of these forms.

[0123] In embodiments, novel amino acid sequences are provided. The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids that function in a manner similar to naturally occurring amino acids, and non-natural or unnatural amino acids, which may be referred to herein as amino acid analogs and amino acid mimetics. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and a functional R group. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but still retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acids may be referred to herein by either their name, their commonly known three letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their commonly accepted one-letter codes.

[0124] An "amino- or carboxy-terminal modification group" refers to any molecule that can be attached to a terminal amine group or terminal carboxy group, respectively. By way of example, such a terminal amine group or terminal carboxy group can be at the end of a polymer molecule, including, but not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example only, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups can be used to improve the therapeutic properties of the polymer molecule, including, but not limited to, increasing the serum half-life of a peptide, polypeptide, or protein.

[0125] In some embodiments, the present disclosure provides novel antibodies and antibody variants. The term "antibody" herein refers to a protein composed of one or more polypeptides substantially encoded by all or part of antibody genes. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. The term "antibody" herein also refers to full-length antibodies and antibody fragments, including naturally occurring antibodies in any organism, antibody variants, and engineered antibodies and antibody fragments. The term "antibody" herein also refers to intact antibodies, monoclonal antibodies, or polyclonal antibodies. The term "antibody" herein also encompasses multispecific and / or bispecific antibodies. The antibodies of the present disclosure include human antibodies. Human antibodies are typically made from two light chains and two heavy chains, each containing a variable region and a constant region. The light chain variable region contains three CDRs, identified herein as CDRL1, CDRL2, and CDRL3, flanked by framework regions. The heavy chain variable region comprises three CDRs, identified herein as CDRH1, CDRH2 and CDRH3, flanked by framework regions.

[0126] The term "antibody fragment" as used herein refers to any form of an antibody other than the full-length form. Antibody fragments, as used herein, include antibodies that are smaller components present within a full-length antibody, as well as engineered antibodies, such as antibody variants. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, Annu. Rev. Biomed. Eng. 2:339-76, 2000; Hudson, Curr. Opin. Biotechnol. 9:395-402, 1998). Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of immunoglobulin heavy and light chains covalently linked by a peptide linker (Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise noted, the description and claims using the terms "antibody" or "antibodies" specifically include "antibody fragment" and "antibody fragments."

[0127] In embodiments, novel antibody-drug conjugates (ADCs) are disclosed. The term "antibody-drug conjugate" or "ADC," as used herein, refers to an antibody molecule, or fragment thereof, covalently linked to one or more biologically active molecules. The biologically active molecules can be conjugated to the antibody via a linker, polymer, or other covalent bond. ADCs are a powerful class of therapeutic constructs that enable targeted delivery of cytotoxic drugs to target cells, such as cancer cells. Due to their targeting function, these compounds exhibit a much higher therapeutic index compared to the same systemic delivery agent. ADCs are developed as intact antibodies or antibody fragments, such as scFvs. The antibody or fragment is linked to one or more copies of the drug via a linker that is stable under physiological conditions but can be cleaved once inside the target cell.

[0128] The term "antigen-binding fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H1 (iv) a single arm V of an antibody, consisting of an Fd fragment L and V H Fv fragment, consisting of domains (v)V H (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which is composed of, for example, V, with or without additional sequences (linker, framework regions, etc.) H(v) a combination of 2 to 6 isolated CDRs, with or without additional sequences (linker, framework region, etc.). Furthermore, the two domains of the Fv fragment, V, L and V H are encoded by separate genes, but using recombinant methods, V L and V H The regions can be linked by a linker that allows them to be produced as a single polypeptide chain that pairs to form a monovalent molecule (also known as single-chain Fv (scFv), see, e.g., Bird et al., Science 242:423-426, 1988) and (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Furthermore, antigen-binding fragments include binding domain immunoglobulin fusion proteins comprising: (i) a binding domain polypeptide (e.g., a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The hinge region may be modified by replacing one or more cysteine ​​residues with serine residues to prevent dimerization. Such binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0129] A typical antigen-binding site consists of a variable region formed by the pairing of a light immunoglobulin chain and a heavy immunoglobulin chain. The structures of antibody variable regions are highly consistent and exhibit very similar structures. These variable regions typically consist of relatively homologous framework regions (FRs) interspersed with three hypervariable regions called complementarity-determining regions (CDRs). The overall binding activity of an antigen-binding fragment is often determined by the sequences of the CDRs. The FRs often play a role in the proper three-dimensional positioning and alignment of the CDRs for optimal antigen binding. Indeed, because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies exhibiting the characteristics of a particular naturally occurring antibody by constructing an expression vector containing CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., Nature 332:323-327, 1998; Jones, P. et al., Nature 321:522-525, 1986; and Queen, C. et al., Proc. Natl. Acad. USA 86:10029-10033, 1989). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation. Germline gene sequences also differ from the sequences of high-affinity secondary repertoire antibodies, which contain mutations throughout the variable genes but are typically clustered in the CDRs. For example, somatic mutations are relatively rare in the amino-terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody to regenerate an intact recombinant antibody with binding properties similar to those of the original antibody. Partial heavy and light chain sequences spanning the CDR regions are typically sufficient for this purpose.Partial sequences are used to determine which germline variable segments and joining gene segments contributed to the recombinant antibody variable genes. The germline sequences are then used to fill in missing portions of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to create a completely synthetic variable region clone. This process has certain advantages, such as the elimination or inclusion of certain restriction sites or the optimization of certain codons. Of course, all or part of the framework regions of the antibodies described herein can be used in conjunction with the CDRs to optimize antibody affinity, specificity, or any other desired property.

[0130] In some embodiments, the present disclosure relates to polymers, such as bifunctional polymers. A "bifunctional polymer," also known as a "bifunctional linker," refers to a polymer containing two functional groups that can specifically react with other moieties to form covalent or non-covalent bonds. Such moieties may include, but are not limited to, natural or unnatural amino acids, or side chains on peptides containing such natural or unnatural amino acids. The other moieties that may be linked to the bifunctional linker or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional linker may have a functional group that is reactive with a group on a first peptide and another functional group that is reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 0188256, U.S. Patent Nos. 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference in their entireties. A "multifunctional polymer," also referred to as a "multifunctional linker," refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, side chains with natural or unnatural amino acids, or peptides containing such natural or unnatural amino acids (including, but not limited to, amino acid side chain groups), to form covalent or non-covalent bonds. Bifunctional or multifunctional polymers may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and the molecule to which it binds or is attached to a compound.

[0131] The term "bioavailability," as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at a site of action or in the systemic circulation. An increase in bioavailability refers to increasing the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at a site of action or in the systemic circulation. By way of example, an increase in bioavailability may be shown as an increase in the concentration of a substance or its active moiety in the blood when compared to other substances or active moieties.

[0132] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active agent" refer to any substance capable of affecting any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with an organism, including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, biologically active molecules include, but are not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or for otherwise enhancing the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal and non-steroidal drugs, microbial toxins, and the like.

[0133] "Modulating biological activity" means increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or enhancing or decreasing the substrate preference of a polypeptide. Analysis of altered biological activity can be performed by comparing the biological activity of the non-naturally occurring polypeptide with that of the naturally occurring polypeptide.

[0134] In some embodiments, the present disclosure relates to amino acids that are biosynthetically incorporated into antibodies. The term "biosynthetically," as used herein, refers to any method that utilizes a translation system (cellular or non-cellular) that includes the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomes. By way of example, unnatural amino acids can be "biosynthetically incorporated" into unnatural amino acid polypeptides using methods and techniques described herein, as are well known in the art. See, e.g., WO 2010 / 011735 and WO 2005 / 074650.

[0135] The term "conservatively modified variants" applies to both natural and non-natural amino acids and natural and non-natural nucleic acid sequences, as well as combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to those natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences, or, if the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, to essentially identical sequences. By way of example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Thus, by way of example, every natural or non-natural nucleic acid sequence herein that encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon in a natural or unnatural nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be altered to obtain a functionally identical molecule. Thus, each silent variation of natural and unnatural nucleic acids that encode natural and unnatural polypeptides is implicit in each described sequence. With respect to amino acid sequences, individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single natural or unnatural amino acid, or a small percentage of natural and unnatural amino acids in the encoded sequence, are "conservatively modified variants" in which the mutation results in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural or unnatural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art.The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co.; 2nd edition, 1993). Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the compositions described herein.

[0136] The term "drug" as used herein refers to any substance used in the prevention, diagnosis, mitigation, treatment, or cure of a disease or condition such as cancer, including but not limited to oral cancer, colorectal cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, non-small-cell-lung (NSCL) cancer, small-cell lung (SCL) cancer, ovarian cancer, breast cancer including triple-negative breast cancer, prostate cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, glioblastoma cancer, or blood cancers including leukemia, lymphoma, or myeloma.

[0137] The term "drug-to-antibody ratio" ("DAR"), as used herein, refers to the average (median) number of drugs conjugated to an antibody in an antibody-drug conjugate (ADC) composition. The DAR value reflects the homogeneity of the ADC population in the composition and also indicates the amount of "payload" (e.g., drug or drug-linker) that can be loaded onto the antibody and delivered to a target (e.g., a cell or diseased tissue). The DAR can be determined by methods known to those skilled in the art, for example, by LC-MS (see, for example, Tang, Y. et al., Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization and Quality Control, Sci Rep 7, 7763 (2017). doi:10.1038 / s41598-017-08151-2, and Chen, Y. Drug-to-antibody ratio (DAR) by UV / Vis spectroscopy, Methods Mol. Biol., 2013;1045:267-73. doi:10.1007 / 978-1-62703-541-5_16). In a non-limiting example, an ADC can have a population distribution of 20% drug-loaded antibodies, where the drug loading is two drugs per antibody, 25% drug-loaded antibodies, where the drug loading is three drugs per antibody, and 55% drug-loaded antibodies, where the drug loading is four drugs per antibody; therefore, in this example, the DAR is [(0.2×2)+(0.25×3)+(0.55×4)]=3.35.

[0138] The term "effective amount," as used herein, refers to a sufficient quantity of an administered agent, compound, or composition to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, the administered agent, compound, or composition includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, a modified non-amino acid polypeptide, or an antibody or variant thereof. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, modified non-natural amino acid polypeptides, or antibodies or variants thereof can be administered for prophylactic, enhancer, and / or therapeutic treatments. An appropriate "effective" amount in any individual case may be determined using techniques, such as dose escalation studies.

[0139] The terms "enhance" or "enhancing" mean to increase or prolong, either in potency or duration, a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong, either in potency or duration, the effect of the therapeutic agent during the treatment of a disease, disorder, or condition. An "enhancing-effective amount," as used herein, refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, disorder, or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0140] The term "humanized or chimeric antibody" refers to a molecule, typically prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species (e.g., murine) and the remainder of the immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. Generally, a humanized antibody will comprise substantially the entirety of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework residues / regions (FR) are those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized forms of rodent antibodies essentially contain the same CDR sequences of the parent rodent antibody, although certain amino acid substitutions may be included to increase affinity, to increase the stability of the humanized antibody, or for other reasons. However, CDR loop exchange does not uniformly result in antibodies with the same binding characteristics as the original antibody, and changes in framework residues (FRs), residues contained in CDR loop supports, can also be introduced into humanized antibodies to maintain antigen-binding affinity. An antigen-binding site can comprise either a complete variable domain grafted onto a constant domain, or only the complementarity-determining regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen-binding site can be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response against the foreign variable region remains (LoBuglio, AF et al., "Mouse / Human Chimeric Monoclonal Antibody in Man: Kinetics and Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224, 1989). Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to reshape them as closely as possible to human form.The variable regions of both the heavy and light chains are known to contain three complementarity-determining regions (CDRs), which vary in response to the antigen in question and determine binding ability, flanked by four framework regions (FRs), which are relatively conserved in a given species and presumably provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific antigen, the variable region can be "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies is described in Kettleborough, CA et al., "Humanization of a Mouse Monoclonal Antibody by CDR-Grafting: The Importance of Framework Residues on Loop Conformation," Protein Engineering 4:773-3783, 1991; Co, MS et al., "Humanized Antibodies for Antiviral Therapy," Proc. Natl. Acad. Sci. (USA) 88:2869-2873, 1991; Carter, P. et al., "Humanization of an Anti-p185her2 Antibody for Human Cancer Therapy," Proc. Natl. Acad. Sci. (USA) 89:4285-4289, 1992; and Co, MS et al., "Chimeric and Humanized Antibodies with Specificity for the CD33 Antigen," J. Immunol. 148:1149-1154, 1992. In some embodiments, a humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibody). In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0141] The term "identical," as used herein, refers to two or more sequences or subsequences that are the same. Additionally, as used herein, the term "substantially identical" refers to two or more sequences that have a percentage of contiguous units that are the same when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over the designated region. Such percentages describe the "percent identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 contiguous units in length, over a region that is about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical if the amino acid residues are the same, but two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region that is at least about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or, if not specified, over the entire sequence of the peptide sequence. Additionally, by way of example only, two or more polynucleotide sequences are identical if the nucleic acid residues are the same, but two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, if not specified, over the entire sequence of the polynucleotide sequence.

[0142] The term "immunogenicity," as used herein, refers to the antibody response to administration of a therapeutic agent. Immunogenicity to a therapeutic non-natural amino acid polypeptide can be obtained using quantitative and qualitative assays to detect anti-non-natural amino acid polypeptide antibodies in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), luminescent immunoassays (LIA), and fluorescent immunoassays (FIA). Analysis of immunogenicity to a therapeutic non-natural amino acid polypeptide involves comparing the antibody response upon administration of the therapeutic non-natural amino acid polypeptide with the antibody response upon administration of the therapeutic natural amino acid polypeptide.

[0143] The term "isolated," as used herein, refers to the separation and removal of a component of interest from components that are not of interest. An isolated material can be in either a dry or semi-dry state, or in a solution, including, but not limited to, an aqueous solution. An isolated component can be in a homogeneous state, or the isolated component can be part of a pharmaceutical composition that includes additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity can be determined using analytical chemistry techniques, including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography (). Additionally, a component is described herein as being substantially purified if it is isolated and is the predominant species present in a preparation. The term "purified," as used herein, can refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or more. By way of example only, a nucleic acid or protein is "isolated" if it is free of at least some of the cellular components with which it is naturally associated, or if the nucleic acid or protein is concentrated to a level greater than its in vivo or in vitro production concentration. Also, by way of example, a gene is isolated if it is separated from open reading frames that flank the gene and encode proteins other than the gene of interest.

[0144] The term "linkage" or "attachment moiety," as used herein, refers to a bond or chemical moiety formed from a chemical reaction between a functional group of one group, such as a linker of the present disclosure, and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, while such chemical moieties can include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, peptide bonds, oximes, and oligonucleotide linkages. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water for extended periods of time, perhaps even indefinitely, at useful pH values, including, but not limited to, physiological conditions. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers can include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the biologically active agent; such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine with an aldehyde, phosphate ester linkages formed by the reaction of an alcohol with a phosphate group, hydrazone linkages which are the reaction product of a hydrazide with an aldehyde, acetal linkages which are the reaction product of an aldehyde with an alcohol, orthoester linkages which are the reaction product of a formate with an alcohol, peptide linkages formed by an amine group at the terminus of a polymer, such as, but not limited to, PEG, and a carboxyl group of a peptide, and oligonucleotide linkages formed by, but not limited to, a phosphoramidite group at the terminus of a polymer and a 5' hydroxyl group of an oligonucleotide.

[0145] The term "linker," as used herein, refers to any polyvalent group that connects or can connect a first group to at least one other group. Typically, a linker is a divalent or trivalent organic moiety that connects a drug (first group) to a biologically active agent (second group), for example, via a linking or attachment moiety, or connects a drug (first group) to a reactive moiety (second group) that can react with a biologically active agent. The linker can be susceptible to cleavage (cleavable linker), such as acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the drug and at least one other group remain active. Alternatively, the linker can be substantially resistant to cleavage (e.g., a stable linker or a non-cleavable linker).

[0146] In some embodiments, the linker, L, is a divalent or trivalent group comprising or consisting of at least one moiety, each at least one moiety independently selected from a bond, an unsubstituted alkylene, a substituted alkylene, -(alkylene-O) n -, optionally substituted arylene, -O-, -C(O)-, -C(S)-, -N(R w )-, -S(O) 0-2 -, methine (-CH)-, amino acids, peptides, disulfides (-SS-), water soluble polymers, and phosphate-based moieties, and combinations thereof; ware independently H, C1-C8 alkyl, or a bond, and each phosphate-based moiety is independently selected from the group consisting of phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosporamidate, pyrophosporamidate, triphosphoramidate, tetraphosphoramidate, phosphorthioate, and diphosphorthioate. In some embodiments, the phosphate-based moiety is a phosphonate, diphosphonate, tetraphosphate ester, or diphosphorthioate. In some embodiments, the phosphate-based moiety is a diphosphonate. In some embodiments, the diphosphonate moiety is conjugated to an oxygen atom of a drug (e.g., duarcomycin of the present disclosure) to provide a drug-linker comprising a pyrophosphate ester. In some embodiments, the water-soluble polymer is (polyethylene) glycol (PEG) or a modified PEG. In some embodiments, the water-soluble polymer is a polysaccharide. Unless explicitly stated otherwise, no orientation of the linker is implied by the direction in which the formula of the linker group is written. As an example, the formula -C(O)CHCH- represents both -C(O)CHCH- and -CHCHC(O)-. In another example, the formula -C(O)CHCH- represents * -C(O)CH2CH2- and -C(O)CH2CH2- * and wherein: * indicates a point of attachment, e.g., a bond to a drug. In some embodiments, when a selected moiety occurs more than once in the same linker, the two or more occurrences are not adjacent. In some embodiments, the linker is not a bond.

[0147] In some embodiments, the linker is a divalent moiety connecting a first group and a second group. In some other embodiments, the linker is a trivalent moiety connecting a first group, a second group, and a third group. In non-limiting examples, the trivalent moiety is C(H) (i.e., methine) or N. In some other embodiments, the linker is a tetravalent moiety connecting a first group, a second group, and a third group.

[0148] In some embodiments, a linker connects at least a first group and a second group, where the first group is a drug and the second group is a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein comprises at least one unnatural amino acid. In some embodiments, the linker connects the drug to an unnatural amino acid of the biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody. Thus, an antibody connected to a drug via a linker can be an antibody-drug conjugate (ADC), e.g., an ADC of the present disclosure.

[0149] In some other embodiments, the linker connects at least a first group and a second group, where the first group is a drug and the second group is a reactive moiety. In some embodiments, the second group is a reactive moiety that can react with a biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein comprises at least one unnatural amino acid. Thus, in some embodiments, the reactive moiety can react with an unnatural amino acid of the biologically active polypeptide or protein. In some embodiments, the biologically active polypeptide or protein is an antibody.

[0150] In some embodiments, a first linker is connected to a second linker, and the combined linker (composite linker) connects at least a first group and a second group. The composite linkers of the present disclosure can include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linker groups. In a non-limiting example, the first, second, and third linker groups can be joined together to provide a composite linker that can connect a first group (e.g., a drug) to at least one other group, such as a reactive moiety and / or a biologically active polypeptide or protein (e.g., an antibody). In some embodiments, the biologically active polypeptide or protein (e.g., an antibody) comprises an unnatural amino acid.

[0151] In some embodiments, the linker is linear. In other embodiments, the linker is branched.

[0152] In some embodiments, the linker is a phosphate-based linker.

[0153] The term "phosphate-based linker," as used herein, refers to a linker that includes a phosphate-based moiety, where the phosphate-based moiety is a phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosporamidate, pyrophosporamidate, triphosphoramidate, tetraphosphoramidate, phosphorothioate, and / or diphosphorthioate.

[0154] The term "metabolite," as used herein, refers to a derivative of a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when a compound, such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" also refers to a biologically active derivative of a compound, which metabolizes a phosphate bond, including, but not limited to, monophosphate, diphosphate, pyrophosphate, and triphosphate.

[0155] The term "metabolized," as used herein, refers to the totality of processes by which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information regarding metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). By way of example only, metabolic products of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be identified either by administering the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides to a host and analyzing tissue samples from the host, or by incubating the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides with hepatocytes in vitro and analyzing the resulting compounds.

[0156] The term "modified," as used herein, refers to the presence of an alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such an alteration, or modification, can be obtained by post-synthetic modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co- or post-translational modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide.

[0157] A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-natural amino acid" are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," "synthetic amino acid," and various hyphenated and non-hyphenated versions thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into growing polypeptide chains by the translation complex. Examples of non-naturally encoded naturally occurring amino acids include, but are not limited to, N-acetylglucosaminoyl-L-serine, N-acetylglucosaminoyl-L-threonine, and O-phosphotyrosine. Additionally, the term "non-natural amino acid" includes, but is not limited to, amino acids that do not occur in nature and may be obtained synthetically or by modification of non-natural amino acids.

[0158] The term "nucleic acid," as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to: (i) analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides, (ii) oligonucleotide analogs, including, but not limited to, PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.), and (iii) conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences and the sequences set forth. By way of example, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0159] The term "pharmaceutically acceptable," as used herein, refers to a material, including but not limited to, a salt, binder, adjuvant, excipient, carrier, or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0160] In some embodiments, the present disclosure relates to polymers. The term "polymer," as used herein, refers to a molecule composed of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols. Polymers of the present disclosure can be linear or branched polymeric polyether polyols, including, but not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. The polymer can be an activated polymer (e.g., activated PEG) that facilitates conjugation with another group, such as a polypeptide, linker, or drug-linker molecule. The polymer can also terminate with a non-reactive moiety, such as an alkyl (e.g., methyl) or alkoxy (e.g., methoxy) moiety. Other exemplary embodiments are listed in commercial supplier catalogs, such as, for example, Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymers have an average molecular weight of about 0.1 kDa to about 100 kDa. Such polymers include, but are not limited to, polymers having a molecular weight of about 100 Da to about 100,000 Da or more, such as about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, and about It may be from about 100 Da to about 100,000 Da, including, but not limited to, 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da.In some embodiments, the molecular weight of the polymer is from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 10,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is in the range of from about 100 Da to about 10,000 Da. In some embodiments, the molecular weight of the polymer is in the range of from about 100 Da to about 5,000 Da. In some embodiments, the molecular weight of the polymer is in the range of from about 100 Da to about 1,000 Da. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, the PEG is linear PEG. In some embodiments, the PEG is branched PEG. The molecular weight of linear or branched PEG is about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, and the like. The molecular weight of the linear or branched PEG may be from about 1,000 Da to about 100,000 Da, including, but not limited to, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is from about 5,000 Da to about 40,000 Da.In some embodiments, the molecular weight of the linear or branched PEG is about 5,000 Da to about 20,000 Da. In other embodiments, the molecular weight of the linear or branched PEG is about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is about 100 Da to about 10,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is about 100 Da to about 5,000 Da. In some embodiments, the molecular weight of the linear or branched PEG is about 100 Da to about 1,000 Da. In some embodiments, the PEG is linear PEG. In some embodiments, the PEG contains a defined number of repeating (-alkylene-O-) units, such as 2, 4, 6, 8, 10, 12, 14, or more units (e.g., PEG-2, PEG-4, PEG-6, PEG-8, PEG-10, PEG-12, PEG-14). In some embodiments, the PEG is a branched PEG. The term "PEGylating" or "PEGylated" refers to the covalent attachment of a specific moiety to a polyethylene glycol (PEG) molecule. In some embodiments, the moiety can be present in a drug, drug-linker, linker, or polypeptide or protein. In some embodiments, the moiety is a hydroxyl group, carboxylic acid, acyl, or amino group, such as a hydroxyl group, carboxylic acid, acyl, or amino group, present in a drug, drug-linker, linker, or polypeptide. In some embodiments, the hydroxyl group, carboxylic acid, acyl, or amino group is present in an amino acid. In some embodiments, the amino acid having a hydroxyl group, carboxylic acid, acyl, or amino group is a natural or unnatural amino acid present in a polypeptide (e.g., an antibody), linker, or drug-linker. The method can include contacting an isolated polypeptide containing a natural or synthetic amino acid with a water-soluble polymer (e.g., PEG) containing a moiety reactive with a natural or synthetic amino acid, or contacting a drug-linker containing a natural or synthetic amino acid therewith.In a non-limiting example, the method can include contacting an isolated anti-TROP2 polypeptide, an isolated anti-HER2 polypeptide, an isolated anti-CD70 polypeptide, an isolated anti-PSMA polypeptide, an isolated anti-HER3 polypeptide, or an isolated anti-GPC3 polypeptide, each of which comprises a natural or synthetic amino acid, with a water-soluble polymer comprising a moiety reactive with a natural or synthetic amino acid.

[0161] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are unnatural amino acids. Additionally, such "polypeptide," "peptide," and "protein" include amino acid chains of any length, including full-length proteins, including, but not limited to, antibodies, in which the amino acid residues are linked by covalent peptide bonds.

[0162] The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid is translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0163] The term "prodrug" or "pharmaceutically acceptable prodrug," as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, does not abolish the biological activity or properties of the drug, and is relatively non-toxic; i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active or more active species through some process, such as conversion by a metabolic pathway. Some prodrugs have chemical groups present on the prodrug that make it less active and / or confer solubility or some other property to the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. Prodrugs are converted to active drugs in the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties, such as better solubility, enhanced delivery properties, such as specific targeting of particular cells, tissues, organs, or ligands, and improved therapeutic value of the drug. Advantages of such prodrugs include, but are not limited to: (i) ease of administration compared to the parent drug, (ii) the prodrug may be bioavailable by oral administration, whereas the parent drug is not, and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include pharmacologically inactive or reduced-activity derivatives of active drugs. Prodrugs can be designed to regulate the amount of a drug or biologically active molecule that reaches a desired site of action through manipulation of drug properties, such as physicochemical, biopharmaceutical, or pharmacokinetic properties. A non-limiting example of a prodrug is a non-natural amino acid polypeptide that is administered as an ester ("prodrug") to facilitate transport across cell membranes, where water solubility is detrimental to mobility, and then metabolically hydrolyzed to the active entity, a carboxylic acid, once inside the cell, where water solubility is beneficial. Prodrugs can be designed as reversible drug derivatives for use as modifiers to enhance drug transport to site-specific tissues.

[0164] The term "prophylactically effective amount," as used herein, refers to an amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide applied prophylactically to a patient that will relieve to some extent one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the patient's state of health, weight, and the like. It is considered well within the skill of one in the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0165] The term "recombinant host cell," also known as a "host cell," refers to a cell that contains an exogenous polynucleotide; methods used to insert the exogenous polynucleotide into the cell include, but are not limited to, direct uptake, transduction, F-mating, or other methods known in the art for generating recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrative vectors, including, but not limited to, plasmids, or may be integrated into the host genome.

[0166] The term "spacer" or "spacer element," as used herein, refers to an atom or functional group that connects a first group to a second group. In some non-limiting embodiments, the spacer is carbonyl (-C(O)-), -C(O)O-, -C(O)N(R)-, -O-, -S-, -N(R)-, where each R is H or alkyl. In some embodiments, the spacer is a divalent spacer.

[0167] The term "subject," as used herein, refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject may be a mammal, including, but not limited to, a human.

[0168] The term "substantially purified," as used herein, refers to a component of interest that may be substantially or essentially free from other components that normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" if a preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. By way of example only, natural or non-natural amino acid polypeptides may be purified from native cells, or, in the case of recombinantly produced natural or non-natural amino acid polypeptides, from host cells. By way of example, a preparation of natural or non-natural amino acid polypeptides may be "substantially purified" if the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating material. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide can be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells.By way of example, a "substantially purified" natural or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more as determined by suitable methods, including but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0169] The term "therapeutically effective amount," as used herein, refers to the amount of a composition containing at least one unnatural amino acid polypeptide and / or at least one modified unnatural amino acid polypeptide that, when administered to a patient already suffering from a disease, condition, or disorder, is sufficient to cure or at least partially arrest, or alleviate to some extent, one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts can be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0170] The term "toxic," or "toxic moiety," or "toxic group," or "cytotoxic," or "cytotoxic payload," or "payload," or "cytotoxic drug," or "drug," as used herein, refers to a cytotoxic compound capable of causing harm, interference, or death. Toxic moieties include, but are not limited to, drugs that comprise or consist of duocarmycin, or analogs or derivatives thereof.

[0171] The terms "treat," "treated," "treating," or "treatment," as used herein, include alleviating, preventing, attenuating, or ameliorating disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of a symptom, inhibiting a disease or symptom, e.g., halting the onset of a disease or symptom, relieving a disease or condition, causing regression of a disease or condition, alleviating the conditions caused by a disease or condition, or halting the symptoms of a disease or condition. The terms "treat," "treated," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments. The terms "treat," "treated," "treating," or "treatment" can refer to a decrease, reduction, or amelioration of one or more symptoms or conditions or diseases associated with an antigen-related or related cancer. The terms "treat," "treated," "treating," or "treatment" can refer to administering an ADC of the disclosure to a subject in need thereof to reduce, reduce, ameliorate, alter, alleviate, affect, or ameliorate an antigen-related or related cancer or disease or symptom or condition, or a predisposition to a condition. The term "capable of specifically binding" refers to a protein or peptide (e.g., an antibody) binding to a predetermined target substance (e.g., an antigen and / or group of antigens), for example, a target substance expressed on the surface of a cell; therefore, the terms "bind to a target cell" or "bind to a cancer cell" should be understood to refer to a protein or peptide (e.g., an antibody) that binds to a predetermined target substance (e.g., an antigen or antigens) expressed on such a cell. Typically, a protein or peptide (e.g., an antibody) has a specific binding affinity of at least about 1×10 7 M1 and / or binds to a predetermined target substance (e.g., an antigen, antigens, or cells) with an affinity that is at least 2-fold higher than its affinity for binding to a non-specific control substance other than the predetermined target substance or a closely related target substance (e.g., BSA, casein, non-cancer cells).

[0172] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C6 10 Examples of suitable polyols include, but are not limited to, alkoxy or aryloxy derivatives thereof (as described in U.S. Pat. No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives including, but not limited to, methylcellulose and carboxymethylcellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycol and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide], and the like, or mixtures thereof. By way of example only, coupling of such water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in changes including, but not limited to, increased water solubility, increased or modified serum half-life, increased or modified therapeutic half-life compared to the unmodified form, increased bioavailability, modified biological activity, extended circulation time, modified immunogenicity, modified physical association properties including, but not limited to, aggregation and multimerization, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. In addition, such water-soluble polymers may or may not have biological activity of their own.

[0173] As used herein, the term "adjusted serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule and determining the concentration of the molecule in each sample. Correlation of serum concentration with time allows for calculation of the serum half-life. By way of example, an adjusted serum half-life may be an increased serum half-life, which may allow for improved dosing regimens or avoid toxic effects. Such an increase in serum may be at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold. Methods for assessing serum half-life are known in the art and may be used to assess the serum half-life of the antibodies and antibody-drug conjugates of the present disclosure.

[0174] The term "modulated therapeutic half-life," as used herein, refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule compared to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. By way of example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. By way of example, increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the unmodified molecule, or, by way of example only, increased or decreased degradation of the molecule by enzymes such as proteases. Methods for assessing therapeutic half-life are known in the art and can be used to assess the therapeutic half-life of the antibodies and antibody-drug conjugates of the present disclosure.

[0175] Introduction Antibody-based therapeutics have emerged as an important component of therapy for an increasing number of human malignancies in fields such as oncology, immunology, inflammatory and infectious diseases. In most cases, the basis for their therapeutic function is the high degree of specificity and affinity that antibody-based drugs have for their target antigens. Arming monoclonal antibodies with drugs, toxins, or radionuclides is yet another strategy by which monoclonal antibodies can induce therapeutic effects. By combining the exquisite targeting specificity of antibodies with the tumor-killing power of toxic effector molecules, immunoconjugates enable highly sensitive discrimination between targets and normal tissues, thereby resulting in fewer side effects than most conventional chemotherapeutic agents. The toxins utilized can be specifically, stably, and irreversibly conjugated to unique sites on the antibody. This unique conjugation process allows for precise control of the location of the toxin on the antibody as well as the number of toxins conjugated to each antibody. Both of these features are important for controlling the biophysical characteristics and toxicity associated with ADCs. (See, for example, Jackson D. et al. (2014) PLoS ONE 9(11):e83865, and Tian F. et al. (2014) Proc. Natl. Acad. Sci. USA 111(15):1766-1771).

[0176] Currently, ADCs are advancing the field of cancer therapy, with several ADCs targeting various drugs either approved or in clinical trials. However, ADCs face challenges due to their lack of therapeutic index and toxicity. Linker technology for conjugating cytotoxic drugs to antibodies affects the stability of ADCs in the systemic circulation. Release of free drug in the circulation instead of within antigen-expressing cancer cells can lead to loss of ADC efficacy, insufficient killing of immunogenic cancer cells, and increased toxicity. Therefore, there is a need to design stable linkers, such as phosphate-based linkers, for drug and antibody conjugation and selective release within cancer cells.

[0177] ADC antibodies and antibody sequences The present invention provides novel ADCs comprising antibodies, antibody fragments, or variants thereof, engineered to have one or more unnatural amino acids incorporated into the heavy and / or light chain amino acid sequences at any desired position. Additionally, the present invention provides ADCs comprising one or more antibodies, antibody fragments, or variants thereof, engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequences conjugated to a drug via a phosphate-based linker. In some embodiments, the antibody, antibody fragment, or variant thereof is selected from the group consisting of PD-1, PD-L1, PSMA, CD70, CD3, HER2, HER3, TROP2, GPC3, VEGFR, EGFR, c-Met (HGFR), CD19, CD22, CD25 (IL-2R alpha), CD30, CD33, CD37, CD46, CD48, CD56 (NCAM-1), CD71 (Transferrin R), CD74, CD79b, CD123 (IL-3R alpha), CD138 (Syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP3), CD205 (LY75), CD221 (IGF-1R), CD262 (TRAIL-1R), and the like. R2), CD276(B7-H3), Mesothelin, EpCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY2c, NaPi2b, Flt-1 , Flt-3, folate receptor alpha, tissue factor (TF), CA6, MUC1, MUC16 (CA-125), BCMA, SLAMF7 (CS1), TIM1, CanAg, Ckit (CD117), E phA2, Nectin4, SLTRK6, FGFR2, LYPD3 (C4.4a), Cadherin 3, 5T4 (TPBG), STEAP1, PTK7, Ephrin-A4, LIV-1 (SLC39A6 or ZIP6), SLC1A5, TENB2, ETBR, Integrin v3, Crypto, AGS-5 (SLC44A4), LY6E, AXL, LAMP1, LRRC15, TNF-alpha, and MN / CA IX antibodies, antibody fragments, or variants. In some embodiments, the antibody, antibody fragment, or variant thereof is a TROP2 antibody, antibody fragment, or variant.In some embodiments, the antibody, antibody fragment, or variant thereof is a HER2 antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a CD3 antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a PSMA antibody, antibody fragment, or variant. In some embodiments, the antibody, antibody fragment, or variant thereof is a CD70 antibody, antibody fragment, or variant. In other embodiments, the invention provides anti-TROP2 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated into any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the invention provides anti-TROP2 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker. In other embodiments, the present invention provides anti-HER2 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated into any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-HER2 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker. In other embodiments, the present invention provides anti-CD3 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated into any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-CD3 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker.In other embodiments, the present invention provides anti-PSMA ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated into any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-PSMA ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker. In other embodiments, the present invention provides anti-CD70 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated into any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-CD70 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker. In other embodiments, the present invention provides anti-HER3 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-HER3 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker. In other embodiments, the present invention provides anti-GPC3 ADCs comprising antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acids incorporated at any desired position in the heavy and / or light chain amino acid sequence. In some embodiments, the present invention provides anti-GPC3 ADCs comprising one or more antibodies, antibody fragments, or variants thereof engineered to have one or more unnatural amino acid sites specifically incorporated into the heavy and / or light chain amino acid sequence conjugated to a drug via a phosphate-based linker.

[0178] The antibodies or antibody fragments or variants of the present disclosure may be human, humanized, engineered, non-human, and / or chimeric antibodies or antibody fragments. The antibodies or antibody fragments or variants provided herein may comprise two or more amino acid sequences. The first amino acid sequence may comprise a first antibody chain, and the second amino acid sequence may comprise a second antibody chain. The first antibody chain may comprise a first amino acid sequence, and the second antibody chain may comprise a second amino acid sequence. An antibody chain may refer to an antibody heavy chain, an antibody light chain, or a combination of a region or all of an antibody heavy chain and a region or all of an antibody light chain. As a non-limiting example, the antibodies provided herein include a heavy chain or a fragment or variant thereof, and a light chain or a fragment or variant thereof. The two amino acid sequences of an antibody, comprising two antibody chains, may be connected, bonded, or linked by one or more disulfide bonds, chemical linkers, peptide linkers, or combinations thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers mediated by one or more unnatural amino acids. Chemical linkers can include chemical conjugates. Peptide linkers include any amino acid sequence that links two amino acid sequences. Peptide linkers can include 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, or 100 or more amino acids. Peptide linkers can be any portion of an antibody, including antibody domains such as the variable domain, CDR1, CDR2, CDR3, and / or a combination of CDRs (complementarity determining regions). In some embodiments, the heavy and light chains are connected, bonded, or linked, for example, via a peptide linker. In some cases, the heavy and light chains are connected, for example, by one or more disulfide bonds.

[0179] The antibodies, antibody fragments, and antibody variants of the present disclosure may interact with or engage antigens on effector cells. Effector cells can include, but are not limited to, immune cells, genetically modified cells with increased or decreased cytotoxic activity, cells involved in host defense mechanisms, anti-inflammatory cells, leukocytes, lymphocytes, macrophages, erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, mast cells, NK cells, B cells, or T cells. In some embodiments, immune cells can be T cells, such as cytotoxic T cells or natural killer T cells. The antibodies or antibody fragments can interact with receptors on T cells, such as, but not limited to, T cell receptors (TCRs). TCRs can include TCR alpha, TCR beta, TCR gamma, and / or TCR delta or TCR zeta. The antibodies or antibody fragments of the present disclosure can bind to receptors on lymphocytes, dendritic cells, B cells, macrophages, monocytes, neutrophils, and / or NK cells. The antibody or antibody fragment of the present disclosure can bind to a cell surface receptor. The antibody or antibody fragment of the present disclosure can bind to an antigen receptor such as the TROP2 antigen receptor, the HER2 antigen receptor, or the CD70 antigen receptor. The antibody or antibody fragment of the present disclosure can be conjugated to a T cell surface antigen.

[0180] Some cell surface antigens have high overexpression patterns in many tumors, making them excellent targets for the development of ADCs. Accordingly, the present disclosure provides novel anti-TROP2 antibodies, anti-HER2 antibodies, anti-CD3 antibodies, anti-PSMA antibodies, anti-CD70 antibodies, anti-HER3 antibodies, anti-GPC3 antibodies, or their corresponding antibody fragments and antibody-drug conjugates for use as therapeutic agents. Novel anti-TROP2 antibodies, antibody fragments or variants thereof, anti-HER2 antibodies, antibody fragments or variants thereof, anti-CD3 antibodies, antibody fragments or variants thereof, anti-PSMA antibodies, antibody fragments or variants thereof, anti-CD70 antibodies, antibody fragments or variants thereof, and anti-HER3 antibodies, antibody fragments or variants thereof are disclosed herein, each of which contains at least one unnatural or non-naturally encoded amino acid. The present invention provides anti-TROP2 antibodies, antibody fragments or variants thereof, anti-HER2 antibodies, antibody fragments or variants thereof, anti-CD3 antibodies, antibody fragments or variants thereof, anti-PSMA antibodies, antibody fragments or variants thereof, anti-CD70 antibodies, antibody fragments or variants thereof, anti-HER3 antibodies, antibody fragments or variants thereof, and anti-GPC3 antibodies, antibody fragments or variants thereof, each having an unnatural amino acid that facilitates antibody conjugation with a drug or drug-linker.

[0181] The antibodies, antibody fragments, or variants provided in the present disclosure may be human, humanized, engineered, non-human, and / or chimeric antibodies or antibody fragments that bind to the extracellular domain of target antigens, which may be overexpressed in some cancers. Thus, novel antibodies, compositions, and antibody-drug conjugates for the treatment and / or diagnosis of antigen-expressing cancers would be beneficial, including, but not limited to, TROP2-expressing cancers, HER2-expressing cancers, CD3-expressing cancers, PSMA-expressing cancers, CD70-expressing cancers, anti-HER3-expressing cancers, and GPC3-expressing cancers.

[0182] Antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, analogs, isoforms, mimetics, fragments, or hybrids of anti-TROP2, anti-HER2, anti-CD3, anti-PSMA, anti-CD70, anti-HER3, and anti-GPC3. Antibodies, antibody fragments, or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0183] Antibodies comprising unnatural amino acids are also disclosed herein. In certain embodiments, antibodies or antibody fragments or variants include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. In some embodiments, an anti-TROP2, anti-HER2, anti-CD3, anti-PSMA, anti-CD70, or anti-GPC3 antibody or antibody fragment or variant comprises one or more unnatural amino acids.

[0184] Non-limiting examples of antibodies or antibody fragments or variants of the present disclosure include the sequences listed in Tables 1-5.

[0185] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-TROP2 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-TROP2 antibodies, antibody fragments, or variants disclosed herein can be humanized. The anti-TROP2 antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, anti-TROP2 analogs, isoforms, mimetics, fragments, or hybrids. The anti-TROP2 antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. The anti-TROP2 antibodies, antibody fragments, or variants disclosed herein comprise the sequences of SEQ ID NOs: 1-17 (Table 1). The antibody, fragment, or variant of the present disclosure can be an anti-TROP2 antibody, fragment, or variant. In certain embodiments, the anti-TROP2 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 1 to 17. In certain embodiments, the anti-TROP2 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 1 to 17.

[0186] The anti-TROP2 antibody may comprise a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 17. In some embodiments, the anti-TROP2 antibody consists of a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 17. In certain embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of any one of SEQ ID NOs: 1, 2, 5, and 6, and a light chain amino acid sequence of any one of SEQ ID NOs: 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0187] In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 17.

[0188] In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 1 and two light chain amino acid sequences of SEQ ID NO: 17.

[0189] In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 17.

[0190] In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 2 and two light chain amino acid sequences of SEQ ID NO: 17.

[0191] In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 17.

[0192] In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 5 and two light chain amino acid sequences of SEQ ID NO: 17.

[0193] In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 6 and a light chain amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 6 and the light chain amino acid sequence of SEQ ID NO: 17.

[0194] In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 3. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 4. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 7. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 8. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 9. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 10. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 11. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 12. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 13. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 14. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 15. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 6 and two light chain amino acid sequences of SEQ ID NO: 17.

[0195] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD70 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD70 antibodies, antibody fragments, or variants disclosed herein can be humanized. The anti-CD70 antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, anti-CD70 analogs, isoforms, mimetics, fragments, or hybrids. The anti-CD70 antibodies, antibody fragments, or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0196] Anti-CD70 antibodies or antibody fragments or variants of the present disclosure comprise one or more of the sequences of SEQ ID NOs: 18-27 (Table 2). The antibodies, fragments, or variants of the present disclosure can be anti-CD70 antibodies, fragments, or variants. In certain embodiments, the anti-CD70 antibodies comprise heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 18-27. In certain embodiments, the anti-CD70 antibodies consist of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 18-27.

[0197] In some embodiments, the anti-CD70 antibody comprises a heavy chain, wherein the heavy chain contains a variable region having the amino acid sequence of SEQ ID NO: 26, and a light chain, wherein the light chain contains a variable region having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-CD70 antibody comprises two heavy chains, each heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 26, and two light chains, each light chain containing a variable region having the amino acid sequence of SEQ ID NO: 27.

[0198] In some embodiments, the anti-CD70 antibody comprises a heavy chain and / or a light chain amino acid sequence selected from the sequences of SEQ ID NOs: 18-27. In some embodiments, the anti-CD70 antibody consists of a heavy chain and / or a light chain amino acid sequence selected from the sequences of SEQ ID NOs: 18-27. In certain embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 or 20 and a light chain amino acid sequence of any one of SEQ ID NOs: 19, 21, 22, 23, and 24. In certain embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18, 20, or 25 and two light chain amino acid sequences of any one of SEQ ID NOs: 19, 21, 22, 23, and 24.

[0199] In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 24.

[0200] In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18 and two light chain amino acid sequences of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18 and two light chain amino acid sequences of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18 and two light chain amino acid sequences of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18 and two light chain amino acid sequences of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 18 and two light chain amino acid sequences of SEQ ID NO: 24.

[0201] In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 20 and a light chain amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 20 and a light chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 20 and a light chain amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 20 and a light chain amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 20 and a light chain amino acid sequence of SEQ ID NO: 24.

[0202] In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 20 and two light chain amino acid sequences of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 20 and two light chain amino acid sequences of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 20 and two light chain amino acid sequences of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 20 and two light chain amino acid sequences of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 20 and two light chain amino acid sequences of SEQ ID NO: 24.

[0203] In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 24.

[0204] In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 25 and two light chain amino acid sequences of SEQ ID NO: 19. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 25 and two light chain amino acid sequences of SEQ ID NO: 21. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 25 and two light chain amino acid sequences of SEQ ID NO: 22. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 25 and two light chain amino acid sequences of SEQ ID NO: 23. In some embodiments, the anti-CD70 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 25 and two light chain amino acid sequences of SEQ ID NO: 24.

[0205] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-HER2 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-HER2 antibodies or antibody fragments or variants disclosed herein can be humanized. The anti-HER2 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-HER2 analogs, isoforms, mimetics, fragments, or hybrids. The anti-HER2 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0206] The anti-HER2 antibodies or antibody fragments or variants of the present disclosure comprise one or more of the sequences of SEQ ID NOs: 28-31 (Table 3). The antibodies, fragments, or variants of the present disclosure can be anti-HER2 antibodies, fragments, or variants. In certain embodiments, the anti-HER2 antibodies comprise heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 28-31. In certain embodiments, the anti-HER2 antibodies consist of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 28-31.

[0207] The anti-HER2 antibody may comprise a heavy and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 28 to 31. In some embodiments, the anti-HER2 antibody consists of a heavy and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 28 to 31. In certain embodiments, the anti-HER2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 28 or 29 and a light chain amino acid sequence of SEQ ID NO: 30 or 31.

[0208] In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 28 and the light chain amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 28 and the light chain amino acid sequence of SEQ ID NO: 31.

[0209] In some embodiments, the anti-HER2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 28 and two light chain amino acid sequences of SEQ ID NO: 30. In some embodiments, the anti-HER2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 28 and two light chain amino acid sequences of SEQ ID NO: 31.

[0210] In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 31.

[0211] In some embodiments, the anti-HER2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 29 and two light chain amino acid sequences of SEQ ID NO: 30. In some embodiments, the anti-HER2 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 29 and two light chain amino acid sequences of SEQ ID NO: 31.

[0212] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-PSMA antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-PSMA antibodies, antibody fragments, or variants disclosed herein can be humanized. The anti-PSMA antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, anti-PSMA analogs, isoforms, mimetics, fragments, or hybrids. The anti-PSMA antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0213] The anti-PSMA antibodies or antibody fragments or variants of the present disclosure comprise one or more of the sequences of SEQ ID NOs: 32-45 (Table 4). The antibodies, fragments, or variants of the present disclosure can be anti-PSMA antibodies, fragments, or variants. In certain embodiments, the anti-PSMA antibodies comprise heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 39-45. In certain embodiments, the anti-PSMA antibodies consist of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 39-45.

[0214] An anti-PSMA antibody may comprise a heavy and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 39-45. In some embodiments, an anti-PSMA antibody consists of a heavy and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 39-45. In certain embodiments, an anti-PSMA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 39 and a light chain amino acid sequence of any one of SEQ ID NOs: 40, 41, 42, or 43. In certain embodiments, an anti-PSMA antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 39 and two light chain amino acid sequences of any one of SEQ ID NOs: 40, 41, 42, or 43.

[0215] In some embodiments, the anti-PSMA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 39 and a light chain amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-PSMA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 39 and a light chain amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-PSMA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 39 and a light chain amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-PSMA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 39 and a light chain amino acid sequence of SEQ ID NO: 43.

[0216] In some embodiments, the anti-PSMA antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 39 and two light chain amino acid sequences of SEQ ID NO: 40. In some embodiments, the anti-PSMA antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 39 and two light chain amino acid sequences of SEQ ID NO: 41. In some embodiments, the anti-PSMA antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 39 and two light chain amino acid sequences of SEQ ID NO: 42. In some embodiments, the anti-PSMA antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 39 and two light chain amino acid sequences of SEQ ID NO: 43.

[0217] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-HER3 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-HER3 antibodies or antibody fragments or variants disclosed herein can be humanized. The anti-HER3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-HER3 analogs, isoforms, mimetics, fragments, or hybrids. The anti-HER3 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. The anti-HER3 antibodies or antibody fragments or variants of the present disclosure comprise the sequences of SEQ ID NOs: 46-58 (Table 5). The antibodies, fragments, or variants of the present disclosure can be anti-HER3 antibodies, fragments, or variants. In certain embodiments, the anti-HER3 antibody comprises heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 46 to 58. In certain embodiments, the anti-HER3 antibody consists of heavy and light chain amino acid sequences selected from the sequences of SEQ ID NOs: 46 to 58.

[0218] The anti-HER3 antibody may comprise a heavy chain and / or a light chain amino acid sequence selected from the sequences of SEQ ID NOs: 46 to 58. In some embodiments, the anti-HER3 antibody consists of a heavy chain and / or a light chain amino acid sequence selected from the sequences of SEQ ID NOs: 46 to 58. In certain embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 or 58 and a light chain amino acid sequence of any one of SEQ ID NOs: 47 to 57.

[0219] In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 49. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 56. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46 and a light chain amino acid sequence of SEQ ID NO: 57.

[0220] In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 47. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 48. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 49. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 50. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 51. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 52. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 53. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 54. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 55. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 56. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 46 and two light chain amino acid sequences of SEQ ID NO: 57.

[0221] In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:47. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:48. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:49. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:50. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:51. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:52. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:53. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:58 and a light chain amino acid sequence of SEQ ID NO:54. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 58 and a light chain amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 58 and a light chain amino acid sequence of SEQ ID NO: 56. In some embodiments, the anti-HER3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 58 and a light chain amino acid sequence of SEQ ID NO: 57.

[0222] In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 47. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 48. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 49. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 50. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 51. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 52. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 53. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 54. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 55. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 56. In some embodiments, the anti-HER3 antibody comprises two heavy chain amino acid sequences of SEQ ID NO: 58 and two light chain amino acid sequences of SEQ ID NO: 57.

[0223] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-GPC3 antibodies or their antibody fragments or variants. In certain embodiments, the anti-GPC3 antibodies, antibody fragments, or variants disclosed herein can be humanized. The anti-GPC3 antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, anti-GPC3 analogs, isoforms, mimetics, fragments, or hybrids. The anti-GPC3 antibodies, antibody fragments, or variants disclosed herein include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc.

[0224] In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD3 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD3 antibodies or antibody fragments or variants disclosed herein can be humanized. The anti-CD3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, anti-CD3 analogs, isoforms, mimetics, fragments, or hybrids. The anti-CD3 antibodies or antibody fragments or variants of the present disclosure include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. The anti-CD3 antibodies or antibody fragments or variants of the present disclosure include one or more sequences, for example, as disclosed in WO 2020 / 047176, the entire contents of which are incorporated herein by reference.

[0225] [Table 1-1]

[0226] [Table 1-2]

[0227] [Table 1-3]

[0228] [Table 2-1]

[0229] [Table 2-2]

[0230] [Table 3]

[0231] [Table 4-1]

[0232] [Table 4-2]

[0233] [Table 5-1]

[0234] [Table 5-2]

[0235] [Table 5-3]

[0236] Unnatural amino acids The present disclosure provides antibodies, antibody fragments, or variants comprising at least one unnatural amino acid. The introduction of at least one unnatural amino acid into an antibody allows for the application of conjugation chemistries that involve specific chemical reactions with one or more unnatural amino acids, while not reacting with the 20 commonly occurring amino acids.

[0237] The unnatural amino acid site selection was based on surface exposure / site accessibility within the antibody, with hydrophobic or neutral amino acid sites selected to maintain charge on the antibody. Methods for introducing unnatural amino acids inserted into sites in proteins are described, for example, in WO 2010 / 011735 and WO 2005 / 074650. The present disclosure employs such methodologies and techniques. The unnatural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group on the side chain of the unnatural amino acid has at least one characteristic and / or activity and / or reactivity that is orthogonal to the chemical reactivity of the 20 common, genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) or that is at least orthogonal to the chemical reactivity of a naturally occurring amino acid present in a polypeptide that includes the unnatural amino acid; and (2) the introduced unnatural amino acid has at least one characteristic and / or activity and / or reactivity that is orthogonal to the chemical reactivity of the 20 common, genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). (1) unnatural amino acids are substantially chemically inert relative to the 20 common, genetically encoded amino acids; (2) unnatural amino acids can be stably incorporated into polypeptides, preferably with stability comparable to that of natural amino acids or under typical physiological conditions, and more preferably, such incorporation can occur via an in vivo system; and (3) unnatural amino acids contain an oxime functional group or a functional group that can be converted to an oxime group by reaction with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide containing the unnatural amino acid (unless, of course, such destruction of biological properties is the goal of the modification / conversion), or if the conversion can occur under aqueous conditions at a pH of about 4 to about 8, or if the reactive site on the unnatural amino acid is an electrophilic site. Any number of unnatural amino acids can be introduced into a polypeptide. Unnatural amino acids can also contain protected or masked oximes, or protected or masked groups that can be converted to an oxime group after deprotection of the protected group or unmasking of the masked group.Unnatural amino acids can also contain protected or masked carbonyl or dicarbonyl groups, which can be converted to carbonyl or dicarbonyl groups after deprotection of the protected group or unmasking of the masked group, thereby available to react with hydroxylamine or oxime to form an oxime group. Oxime-based unnatural amino acids can be synthesized by methods well known in the art (see, e.g., WO 2013 / 185117 and WO 2005 / 074650), including (a) reaction of a hydroxylamine-containing unnatural amino acid with a carbonyl- or dicarbonyl-containing reagent, (b) reaction of a carbonyl- or dicarbonyl-containing unnatural amino acid with a hydroxylamine-containing reagent, or (c) reaction of an oxime-containing unnatural amino acid with certain carbonyl- or dicarbonyl-containing reagents.

[0238] In some embodiments, the selection of a non-naturally encoded amino acid site is based on surface exposure. For example, one potential site is an amino acid with a solvent-accessible surface area ratio of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In some embodiments, one potential site is an amino acid with a solvent-accessible surface area ratio of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% or more. The solvent-accessible surface area can be calculated using the DSSP program [Biopolymers, 22, 2577-2637 (1983)] using the crystal structure data file of an antibody or antibody fragment deposited in the Protein Data Bank (PDB).

[0239] The ratio of the solvent-accessible surface area of ​​the amino acid residue of interest can be calculated by dividing the solvent-accessible surface area of ​​the antibody structure calculated above by the solvent-accessible surface area of ​​alanine-X-alanine (where X represents the amino acid residue of interest). In this regard, two or more PDB files may exist for a protein of one species, any one of which can be used in the present invention.

[0240] Alternatively, the solvent accessibility of an amino acid can be determined by a solvent accessibility test in which a functional group (i.e., a thiol, amino, or carbonyl group) on the amino acid is functionalized when treated with an electrophile or a nucleophile, etc. Based on the results of the test, the functional group (i.e., a thiol, amino, or carbonyl group) can be said to be at least 50% solvent accessible if, for example, at least 50% of the functional group is functionalized in the test. In some embodiments, the unnatural amino acid site is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% solvent accessible. Examples of solvent accessibility tests include, but are not limited to, propargylation of surface thiol groups or α-bromopyruvate reacting with surface thiol groups.

[0241] Unnatural amino acids that can be used in the methods and compositions described herein include, but are not limited to, amino acids including amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, glycosylated amino acids such as sugar-substituted serines, other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with elongated side chains compared to natural amino acids, including, but not limited to, polyethers or long-chain hydrocarbons containing, but not limited to, more than about 5 or more than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties.

[0242] In some embodiments, antibodies comprising one or more unnatural amino acids are disclosed herein. The one or more unnatural amino acids can be encoded by a codon that does not encode one of the 20 natural amino acids. The one or more unnatural amino acids can be encoded by a nonsense codon (stop codon). The stop codon can be an amber codon. The amber codon can include a UAG sequence. The stop codon can be an ochre codon. The ochre codon can include a UAA sequence. The stop codon can be an opal or umber codon. The opal or umber codon can include a UGA sequence. The one or more unnatural amino acids can be encoded by a four-base codon.

[0243] Unnatural amino acids of the disclosure include: 1) substituted phenylalanine and tyrosine analogs, such as 4-amino-L-phenylalanine, 4-acetyl-L-phenylalanine, 4-azido-L-phenylalanine, 4-nitro-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-isopropyl-L-phenylalanine, 3-nitro-L-tyrosine, O-methyl-L-tyrosine, and O-phosphotyrosine; 2) amino acids that can be photocrosslinked, such as amino acids with an aryl azide or benzophenone group, such as 4-azidophenylalanine or 4-benzoylphenylalanine; and 3) amino acids with unique chemical reactivity, such as 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-2-propyn-1-yl-L-tyrosine, N-(ethylthio)thiocarbonyl-L-phenylalanine, and p-(3-oxobutanoic acid). yl)-L-phenylalanine, 4) heavy atom-containing amino acids, for example, for phasing in X-ray crystallography, such as, for example, 4-iodo-L-phenylalanine or 4-bromo-L-phenylalanine, 5) redox-active amino acids, for example, 3,4-dihydroxy-L-phenylalanine, 6) fluorinated amino acids, for example, 2-fluorophenylalanine (e.g., 2-fluoro-L-phenylalanine), 3-fluorophenylalanine (e.g., 3-fluoro-L-phenylalanine) or 4-fluorophenylalanine (e.g., 4-fluoro-L-phenylalanine), 7) fluorescent amino acids, for example, amino acids containing naphthyl, dansyl or 7-aminocoumarin side chains, 8) photocleavable or photoisomerizable amino acids, for example, amino acids containing azobenzyl or nitrobenzyl, for example, cysteine, serine or tyrosine containing azobenzyl or nitrobenzyl, 9) β-amino acids (e.g., β 2 or β 3amino acids), 10) homo-amino acids, for example, homoglutamine (e.g., beta-homoglutamine) or homophenylalanine (e.g., beta-homophenylalanine), 11) proline or pyruvate derivatives, 12) 3-substituted alanine derivatives, 14) glycine derivatives, 15) linear core amino acids, 16) diamino acids, 17) D-amino acids, 18) N-methyl amino acids, 19) phosphotyrosine mimetics, for example, carboxymethylphenylalanine (pCMF) (e.g., 4-carboxymethyl-L-phenylalanine), 20) 2-aminooctanoic acid, and 21) amino acids containing a sugar moiety, for example, N-acetyl-L-glucosaminyl-L-serine, beta-N-acetylglucosamine-O-serine, N-acetyl-L-galactosaminyl-L-serine, alpha-N-acetylgalactosamine-O-serine, O-(3-O-galactosamine), and O-(mannosyl)-L-serine, N-acetyl-L-glucosaminyl-L-threonine, alpha-N-acetylgalactosamine-O-threonine, 3-O-(N-acetyl-beta-D-glucosaminyl)-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, N4-(β-N-acetyl-D-glucosaminyl)-L-asparagine, and O-(mannosyl)-L-serine, amino acids in which the naturally occurring N- or O-bond between the amino acid and sugar is replaced by a covalent bond not commonly found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc., or amino acids containing a sugar not commonly found in naturally occurring polypeptides, such as 2-deoxy-glucose, 2-deoxy-galactose, etc.Specific examples of unnatural amino acids include p-acetylphenylalanine (4-acetylphenylalanine) (including 4-acetyl-L-phenylalanine, also referred to herein as p-acetyl-L-phenylalanine (pAF)), 4-boronophenylalanine (pBoF) (e.g., 4-borono-L-phenylalanine, 4-propargyloxyphenylalanine (pPrF) (e.g., 4-propargyloxy-L-phenylalanine), O-methyltyrosine (e.g., For example, O-methyl-L-tyrosine), 3-(2-naphthyl)alanine (NapA) (e.g., 3-(2-naphthyl)-L-alanine), 3-methylphenylalanine (e.g., 3-methyl-L-phenylalanine), O-allyl tyrosine (e.g., O-allyl-L-tyrosine), O-isopropyl tyrosine (e.g., O-isopropyl-L-tyrosine), dopamine (e.g., L-dopa), 4-isopropylphenylalanine (e.g., 4-isopropyl-L-phenylalanine) , 4-azidophenylalanine (pAz) (e.g., 4-azido-L-phenylalanine), 4-benzoylphenylalanine (pBpF) (e.g., 4-benzoyl-L-phenylalanine), O-phosphoserine (e.g., O-phospho-L-serine), O-phosphotyrosine (e.g., O-phospho-L-tyrosine), 4-iodophenylalanine (pIF) (e.g., 4-iodo-L-phenylalanine), 4-bromophenylalanine (e.g., 4-bromo-L-phenylalanine), Examples of suitable phenylalanine derivatives include, but are not limited to, 4-aminophenylalanine (e.g., 4-amino-L-phenylalanine), 4-cyanophenylalanine (pCNF) (e.g., 4-cyano-L-phenylalanine, (8-hydroxyquinolin-3-yl)alanine (HQA) (e.g., (8-hydroxyquinolin-3-yl)-L-alanine), (2,2-bipyridin-5-yl)alanine (BipyA) (e.g., (2,2-bipyridin-5-yl)-L-alanine), and the like.Additional unnatural amino acids are disclosed in Liu et al. (2010) Annu Rev Biochem, 79:413-44, Wang et al. (2005) Angew Chem Int Ed, 44:34-66, and published WO 2012 / 166560, WO 2012 / 166559, WO 2011 / 028195, WO 2010 / 037062, WO 2008 / 083346, WO 2008 / 077079, WO 2007 / 094916, WO 2007 / 079130, WO 2007 / 070659, and WO 2007 / 059312, the entire contents of each of which are incorporated herein by reference. In some embodiments, one or more unnatural amino acids can be p-acetylphenylalanine. In some more particular embodiments, one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF).

[0244] In some embodiments, the one or more unnatural amino acids are 4-acetylphenylalanine, 3-O-(N-acetyl-beta-D-glucosaminyl)threonine, N4-(β-N-acetyl-D-glucosaminyl)asparagine, O-allyltyrosine, alpha-N-acetylgalactosamine-O-serine, alpha-N-acetylgalactosamine-O-threonine, 2-aminooctanoic acid, 2-aminophenylalanine, 3-aminophenylalanine, 4-aminophenylalanine, 2-aminotyrosine, 3-allyl Aminotyrosine, 4-azidophenylalanine, 4-benzoylphenylalanine, (2,2-bipyridin-5yl)alanine, 3-boronophenylalanine, 4-boronophenylalanine, 4-bromophenylalanine, p-carboxymethylphenylalanine, 4-carboxyphenylalanine, p-cyanophenylalanine, 3,4-dihydroxyphenylalanine, 4-ethynylphenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, O-(3 -OD-Galactosyl-N-acetyl-beta-D-galactosaminyl)serine, homoglutamine, (8-hydroxyquinolin-3-yl)alanine, 4-iodophenylalanine, 4-isopropylphenylalanine, Oi-propyltyrosine, 3-isopropyltyrosine, O-mannopyranosylserine, 2-methoxyphenylalanine, 3-methoxyphenylalanine, 4-methoxyphenylalanine, 3-methylphenylalanine, O-methyltyrosine, 3-(2-naphthyl)alanine, 5-nitrohistidine 4-nitrophenylalanine, 4-nitrohistidine, 4-nitroleucine, 2-nitrophenylalanine, 3-nitrophenylalanine, 4-nitrophenylalanine, 4-nitrotryptophan, 5-nitrotryptophan, 6-nitrotryptophan, 7-nitrotryptophan, 2-nitrotyrosine, 3-nitrotyrosine, O-phosphoserine, O-phosphotyrosine, 4-propargyloxyphenylalanine, O-2-propyn-1-yltyrosine, 4-sulfophenylalanine, and O-sulfotyrosine.

[0245] In some further embodiments, the one or more unnatural amino acids are 4-acetyl-L-phenylalanine (para-acetyl-L-phenylalanine (pAF)), 3-O-(N-acetyl-β-D-glucosaminyl)-L-threonine, N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine, O-allyl-L-tyrosine, alpha-N-acetylgalactosamine-OL-serine, alpha-N-acetylgalactosamine-OL-threonine, 2-aminooctanoic acid, 2-amino-L-phenylalanine ... , 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2,2-bipyridin-5-yl)-L-alanine, 3-borono-L-phenylalanine, 4-borono-L-phenylalanine, 4-bromo-L-phenylalanine, p-carboxymethyl-L-phenylalanine, 4-carboxy-L-phenylalanine, p-cyano-L-phenylalanine, 3,4-Dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine, O-(3-OD-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine, L-homoglutamine, (8-hydroxyquinolin-3-yl)-L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, Oi-propyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine, 3-methyl-L-phenylalanine The amino acid sequence is selected from the group consisting of 5-nitro-L-alanine, O-methyl-L-tyrosine, 3-(2-naphthyl)-L-alanine, 5-nitro-L-histidine, 4-nitro-L-histidine, 4-nitro-L-leucine, 2-nitro-L-phenylalanine, 3-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propargyloxy-L-phenylalanine, O-2-propyn-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine and O-sulfo-L-tyrosine. In some embodiments, one or more unnatural amino acids can be p-acetyl-L-phenylalanine (pAF). Thus, in some embodiments, each and every one or more unnatural amino acids is pAF.

[0246] In certain embodiments of the present disclosure, antibodies with at least one unnatural amino acid comprise at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises the attachment of a molecule comprising a second reactive group to at least one unnatural amino acid comprising a first reactive group, including, but not limited to, a water-soluble polymer, a derivative of polyethylene glycol, a drug, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a biologically active agent, a small molecule, or any combination of the above, or any other desired compound or substance, using chemical methodology known to those of skill in the art to be suitable for the particular reactive group. For example, the first reactive group is an alkynyl moiety (including, but not limited to, the unnatural amino acid p-propargyloxyphenylalanine, where the propargyl group is sometimes referred to as an acetylene moiety) and the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry is utilized. In another example, the first reactive group is an azido moiety (including, but not limited to, the unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is an alkynyl moiety. Certain embodiments of the modified antibody polypeptides of the present disclosure use at least one unnatural amino acid (including, but not limited to, an unnatural amino acid comprising a keto functionality) that comprises at least one post-translational modification, wherein the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification occurs in vivo, in a eukaryotic cell or a non-eukaryotic cell. In other embodiments, the post-translational modification occurs in vitro. In other embodiments, the post-translational modification occurs in vitro and in vivo.

[0247] In some embodiments, unnatural amino acids can be modified to incorporate a chemical group. In some embodiments, unnatural amino acids can be modified to incorporate a ketone group. One or more unnatural amino acids can include at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or a combination thereof. One or more unnatural amino acids can include at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl / alkyl azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine functional group, or a combination thereof.

[0248] In some embodiments disclosed herein, unnatural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. In some embodiments, unnatural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. Methods for incorporating unnatural amino acids into molecules, such as proteins, polypeptides, or peptides, are disclosed in U.S. Patent Nos. 7,332,571, 7,928,163, 7,696,312, 8,008,456, 8,048,988, 8,809,511, 8,859,802, 8,791,231, 8,476,411, or 9,637,411 (each of which is incorporated by reference herein in its entirety), and in the Examples herein. One or more unnatural amino acids can be incorporated by methods known in the art. For example, cell-based or cell-free systems can be used, and auxotrophic strains can also be used in place of engineered tRNAs and synthetases. In certain embodiments, orthogonal tRNA synthetases are disclosed in, for example, WO2002085923(A2), WO2002086075(A2), WO2004035743(A2), WO2007021297(A1), WO2006068802(A2), and WO2006069246(A2), the entire contents of which are incorporated herein by reference. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in the antibody, antibody fragment, or variant may involve mutating one or more nucleotides in the nucleotide sequence encoding the antibody, antibody fragment, or variant. Mutating one or more nucleotides in the nucleotide sequence encoding the antibody, antibody fragment, or variant may involve changing a codon encoding an amino acid to a nonsense codon.Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include modifying one or more amino acid residues in the antibody or antibody fragment or variant to generate one or more amber codons in the antibody or antibody fragment or variant. One or more unnatural amino acids can be incorporated into the antibody or antibody fragment or variant in response to an amber codon. One or more unnatural amino acids can be site-specifically incorporated into the antibody or antibody fragment or variant. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include one or more genetically encoded unnatural amino acids with orthogonal chemical reactivity to the standard 20 amino acids for site-specific modification of a biologically active molecule or targeting agent. Incorporating one or more unnatural amino acids can include using a tRNA / aminoacyl-tRNA synthetase pair to site-specifically incorporate one or more unnatural amino acids into a defined site in a biologically active molecule or targeting agent in response to one or more amber nonsense codons. Additional methods for incorporating unnatural amino acids include, but are not limited to, those disclosed in Chatterjee et al., A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli, Biochemistry, 2013; Kazane et al., J Am Chem Soc, 135(1):340-6, 2013; Kim et al., J Am Chem Soc, 134(24):9918-21, 2012; Johnson et al., Nat Chem Biol, 7(11):779-86, 2011; and Hutchins et al., J Mol Biol, 406(4):595-603, 2011. One or more unnatural amino acids can be produced through selective reaction of one or more natural amino acids. The selective reaction can be mediated by one or more enzymes.In a non-limiting example, selective reaction of one or more cysteines with a formylglycine generating enzyme (FGE) can produce one or more formylglycines, as described in Rabuka et al., Nature Protocols 7:1052-1067, 2012. One or more unnatural amino acids can include a linker-forming chemical reaction. The linker-forming chemical reaction can include a bioorthogonal reaction. The linker-forming chemical reaction can include click chemistry. See, e.g., WO 2006 / 050262, incorporated herein by reference in its entirety.

[0249] Any position in an antibody or antibody fragment is suitable for selection for incorporating an unnatural amino acid, and selection can be based on rational design or by random selection for any or specific desired purpose. Selection of desired sites can be based on generating a unnatural amino acid polypeptide (which may be further modified or left unmodified) with any desired property or activity, including, but not limited to, receptor binding modulator, receptor activity modulator, modulator of binding with a binding partner, modulator of binding partner activity, modulator of binding partner structure, dimer or multimer formation, no change in activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide, such as solubility, aggregation, or stability. Alternatively, sites identified as important for biological activity may also be good candidates for substitution with an unnatural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make serial substitutions with an unnatural amino acid at each position along the polypeptide chain and observe the effect on the activity of the polypeptide. Any means, technique, or method for selecting the location of a non-natural amino acid substitution into any polypeptide is suitable for use in the methods, techniques, and compositions described herein.

[0250] The structure and activity of naturally occurring variants of a polypeptide, including deletions, can also be investigated to determine regions of the protein that are likely to tolerate substitution with an unnatural amino acid. Once residues that are likely to be intolerant to substitution with an unnatural amino acid have been eliminated, the impact of proposed substitutions at each remaining position can be examined using methods including, but not limited to, the three-dimensional structure of the relevant polypeptide and any associated ligands or binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Structure Bank (PDB, see the World Wide Web at rcsb.org), a centralized database containing three-dimensional structural data for large protein and nucleic acid molecules, and can be used to identify amino acid positions that can be substituted with an unnatural amino acid. In addition, if three-dimensional structural data is not available, models can be generated that explore the secondary and tertiary structure of the polypeptide. Thus, the identity of amino acid positions that can be substituted with an unnatural amino acid can be determined by one of skill in the art.

[0251] Examples of sites for incorporation of unnatural amino acids include, but are not limited to, regions for binding to binding proteins or ligands that are excluded from potential receptor binding regions or that may be fully or partially solvent exposed, have minimal or no hydrogen bonding interactions with nearby residues, be minimally exposed to nearby reactive residues, and / or be in regions that are highly flexible as predicted by the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand, or binding protein.

[0252] A wide variety of unnatural amino acids can be substituted into or incorporated into a given position in a polypeptide. By way of example, a particular unnatural amino acid can be selected for incorporation based on its preference for conservative substitutions, which is determined by examining the three-dimensional crystal structure of the polypeptide with its associated ligand, receptor, and / or binding protein.

[0253] Linker In some aspects, the present disclosure relates to a linker for intracellular delivery of drug conjugates. Many procedures and linker molecules for conjugating various compounds to peptides are known. For example, see European Patent Application No. 0188256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, 4,569,789 and 10,550,190, PCT Publication No. WO2012 / 166559(A1), ... See U.S. Patent Application Publication No. WO2013 / 185117(A1), WO2013 / 192360(A1) and WO2022 / 040596(A1), and U.S. Patent Application Publication No. US2017 / 0182181(A1), the contents of each of which are incorporated herein by reference in their entirety.

[0254] In some embodiments, the present disclosure relates to phosphate-based linkers for intracellular delivery of drug conjugates (see, e.g., U.S. Pat. No. 10,550,190). The phosphate-based linkers of the present disclosure comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group (a phosphate group) and a linker arm, and optionally a spacer. The drug-linker can be covalently attached to a reactive functional group that can be covalently attached to a cell-specific targeting ligand, such as an antibody or antibody fragment. The phosphate-based linkers have differential, tunable stability in blood compared to the intracellular environment (e.g., the lysosomal compartment). Thus, ADCs containing these phosphate-based linkers are stable in circulation (plasma / blood) but reactive or cleavable in the intracellular compartment (lysosome), making them useful for intracellular delivery of drug conjugates. The phosphate-based linker can be conjugated to a drug, and the reactive functional group can be conjugated to a cell-specific targeting ligand, such as an anti-TROP2 antibody, an anti-HER2 antibody, an anti-CD70 antibody, an anti-CD3 antibody, or an anti-PSMA antibody. The phosphate-based linkers of the present disclosure are designed to engineer ADCs such that the likelihood of the conjugate forming aggregates is reduced compared to conjugates in which the same drug is conjugated to an antibody or targeting ligand using a linker that is not a phosphate-based linker. Furthermore, the phosphate-based linker design, stability, pH, redox sensitivity, and protease susceptibility affect the circulation stability and release of the drug.

[0255] Methods for selecting and designing linkers are well known in the art. Linkers can be designed ab initio, by way of example only, as part of a high-throughput screening process (where large numbers of polypeptides can be designed, synthesized, characterized, and / or tested) or based on the researcher's goals. Linkers can also be designed based on the structure of a known or partially characterized polypeptide. Principles for selecting which amino acids to replace and / or modify, as well as the selection of modifications to use, are described, for example, in WO 2013 / 185117. Linkers can be designed to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic efficacy of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications.

[0256] Generally, the linkers of the present disclosure can be units that can be combined with one or more additional units such that the combined linker unit can bind to one or more drugs. Each linker unit can be composed of one or more moieties, each of which can appear one or more times.

[0257] In some embodiments, a linker of the present disclosure comprises at least one phosphate-based moiety, as disclosed herein. In some embodiments, a linker comprises a phosphate-based moiety and further comprises at least one moiety or unit that is not phosphate-based. In some embodiments, a linker is a bivalent linker. In some embodiments, a linker is a trivalent linker. In some embodiments, a linker is a tetravalent linker.

[0258] Thus, in some embodiments, the present disclosure provides a phosphate-based linker. The phosphate-based linker of the present disclosure or the drug-linker of the present disclosure can include a phosphate-based moiety, wherein the phosphate-based moiety is a phosphate ester, a pyrophosphate ester, a triphosphate ester, a tetraphosphate ester, a phosphonate, a diphosphonate, a phosphoramidate, a pyrophosphoramidate, a triphosphoramidate, a tetraphosphoramidate, a phosphorothioate, and / or a diphosphorthioate. Thus, the phosphate-based linker or drug-linker of the present disclosure can be: structure

[0259] [ka] a phosphate ester having the formula structure

[0260] [ka] a phosphonate having the formula structure

[0261] [ka] a pyrophosphate ester having the formula structure

[0262] [ka] a diphosphonate having structure

[0263] [ka] a triphosphate ester having the formula structure

[0264] [ka] Tetraphosphate ester having: structure

[0265] [ka] a phosphorothioate having the formula structure

[0266] [ka] diphosphorthioates having the formula structure

[0267] [ka] a phosphoramidate having the formula structure

[0268] [ka] a pyrophosphoramidate having the formula structure

[0269] [ka] and / or a triphosphoramidate having structure

[0270] [ka] The invention can include tetraphosphoramidates having the formula:

[0271] In some embodiments, a phosphate-based linker of the present disclosure comprises a phosphate-based moiety selected from the group consisting of pyrophosphate esters and diphosphonates. In some embodiments, a drug-linker of the present disclosure comprises a phosphate-based moiety selected from the group consisting of pyrophosphate esters and diphosphonates.

[0272] In some embodiments, a phosphate-based linker, or drug-linker, of the present disclosure comprises a pyrophosphate ester. For example, in some embodiments, a drug (e.g., a duocarmycin compound of the present disclosure) comprises an oxygen atom (—O—) linked to the phosphorus atom of the diphosphonate moiety, thereby providing a drug-linker comprising a pyrophosphate ester.

[0273] In some other embodiments, the phosphate-based linker, or drug-linker, of the present disclosure comprises a diphosphonate.

[0274] In some embodiments, the phosphate-based linker is a bivalent linker.

[0275] In some embodiments, the phosphate-based moiety is covalently bonded via the phosphorus atom of the phosphate-based moiety to the —O— atom of a drug, e.g., a compound of Formula (X), or Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic), or Formula (Id), or Formula (IL), or Formula (ILa), or Formula (ILb), or Formula (ILc), or Formula (ILd), or an ADC of Formula (II) disclosed herein.

[0276] In some embodiments, the phosphate-based linker further comprises at least one additional moiety. In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, -(alkylene-O)-, optionally substituted arylene, -O-, -C(O)-, -N(R w )-, -S(O) 0-2 -, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkylalkynyl.w is independently H or unsubstituted C1-C8 alkyl. In yet some further embodiments, each R w is independently H or methyl.

[0277] In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R w )-, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkylalkynyl. w is independently H or unsubstituted C1-C8 alkyl. In yet some further embodiments, each R w is independently H or methyl.

[0278] It should be understood that each at least one additional moiety that can be present in a phosphate-based linker of the present disclosure can occur one or more times within the linker. In a non-limiting example, a phosphate-based linker of the present disclosure can include one or more unsubstituted alkylene groups, and each unsubstituted alkylene group can be the same or different. In another non-limiting example, a phosphate-based linker of the present disclosure can include one or more amino acids, and each amino acid can be the same or different.

[0279] In some embodiments, the linker comprises at least one alkylene group.

[0280] In some embodiments, the linker comprises an amino acid, in some embodiments, the amino acid is serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N eIn some embodiments, the amino acid is selected from the group consisting of lysine or N-methyl-lysine. e In some embodiments, the amino acid is N-methyl-lysine. In some embodiments, the amino acid is lysine. e -methyl-lysine.

[0281] In some embodiments, the linker comprises a water-soluble polymer.

[0282] In some embodiments, the linker comprises a water-soluble polymer and an amino acid, and the water-soluble polymer is conjugated to the amino acid. In some embodiments, the water-soluble polymer is conjugated to a side chain of the amino acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

[0283] In some embodiments, a phosphate-based linker of the present disclosure comprises a water-soluble polymer and an amino acid that is serine, threonine, or tyrosine, where the water-soluble polymer is conjugated to the side chain —OH group of the serine, threonine, or tyrosine. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

[0284] In some embodiments, a phosphate-based linker of the present disclosure comprises a water-soluble polymer and an amino acid that is cysteine, wherein the water-soluble polymer is conjugated to the side chain -SH group of the cysteine. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

[0285] In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and an amino acid that is aspartic acid or glutamic acid, where the water-soluble polymer is conjugated to the side chain carboxylate group of the aspartic acid or glutamic acid. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

[0286] In some embodiments, the phosphate-based linkers of the present disclosure comprise a water-soluble polymer and a lysine and N e and an amino acid which is -N-methyl-lysine, and the water soluble polymer is lysine or -N e -methyl-lysine side chain -N e H(R) groups, where R is independently H or methyl. In some embodiments, the water-soluble polymer is conjugated to the amino acid via a spacer element.

[0287] In some embodiments, the water-soluble polymer is a polysaccharide.

[0288] In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0289] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0290] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0291] In some embodiments, the phosphate-based linker of the present disclosure is a linker selected from the group of linkers listed in Table 6.

[0292] [Table 6-1]

[0293] [Table 6-2]

[0294] In some embodiments, each i in Table 6 is 1. In some other embodiments, each i in Table 6 is 0.

[0295] In some embodiments, each U is independently optionally substituted with a water-soluble polymer.

[0296] In some embodiments, each n in Table 6 is independently an integer from 1 to 10. In some embodiments, each n in Table 6 is independently 1, 2, or 3.

[0297] In some embodiments, each alkylene in Table 6 is independently —(CH 2 )—, —(CH 2 ) 2 —, or —(CH 2 ) 3 —.

[0298] In some embodiments, each linker in Table 6 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 6 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 6 is substituted with one water soluble polymer.

[0299] In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U. In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U via a spacer element.

[0300] In some embodiments, the water-soluble polymer is a polysaccharide.

[0301] In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0302] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0303] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0304] Unless explicitly stated otherwise, it is understood that no orientation of the linker is implied by the direction in which the formula of the linker group is written. By way of example only, a group of the formula -alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -teeth, -Alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -and-(O) i In another example, the formula -alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -teeth, * -Alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i - and - alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i - * and wherein: * indicates a connection point, for example, a connection to a drug.

[0305] It is also understood that independent selections can be made within a given linker, where each alkylene (or other variable) in the linker is independently selected from a group of variables. * -Alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -Alkylene-(O-alkylene) n - (wherein each alkylene is independently -(CH2)-, -(CH2)2-, or -(CH2)3-) is selected from the following species: * -(CH2)-OP(=O)(OH)-OP(=O)(OH)-(O) i -(CH2)2-(O-(CH2)3) n -, * -(CH2)2-OP(=O)(OH)-OP(=O)(OH)-(O) i -(CH2)3-(O-CH2) n -, * -(CH2)2-OP(=O)(OH)-OP(=O)(OH)-(O)i -(CH2)2-(O-(CH2)2) n -, * -(CH2)3-OP(=O)(OH)-OP(=O)(OH)-(O) i -(CH2)-(O-(CH2)2) n -and * -(CH2)2-OP(=O)(OH)-OP(=O)(OH)-(O) i -(CH2)-(O-(CH2)2) n - Including, but not limited to:

[0306] Further, by way of example only, * -Alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -Alkylene-(O-alkylene) n wherein each alkylene is independently —(CH)—, —(CH)—, or —(CH)— * -Alkylene-OP(=O)(OH)-OP(=O)(OH)-(O) i -Alkylene'-(O-alkylene'') n -, and each alkylene, alkylene', and alkylene'' is independently -(CH2)-, -(CH2)2-, or -(CH2)3-. Similarly, * -(Alkylene-O) n -P(=O)(OH)-OP(=O)(OH)-(O) i -(Alkylene-O) n -J-Alkylene-(Alkylene-O) n - (wherein each alkylene is independently -(CH2)-, -(CH2)2-, or -(CH2)3-, and each n is independently 1, 2, or 3) is * -(Alkylene-O) n -P(=O)(OH)-OP(=O)(OH)-(O) i -(Alkylene'-O) n’ -J-Alkylene''-(Alkylene''-O) n’’where each alkylene', alkylene''', and alkylene''' is independently (CH)-, -(CH)-, or -(CH)-; and each n, n', and n'' is independently 1, 2, or 3.

[0307] In some embodiments, the phosphate-based linker of the present disclosure is a linker selected from the group of linkers listed in Table 7.

[0308] [Table 7]

[0309] In some embodiments, each i in Table 7 is 1. In some other embodiments, each i in Table 7 is 0.

[0310] In some embodiments, each U is independently optionally substituted with a water-soluble polymer.

[0311] In some embodiments, each n in Table 7 is independently an integer from 1 to 10. In some embodiments, each n in Table 7 is independently 1, 2, or 3.

[0312] In some embodiments, each alkylene in Table 7 is independently —(CH 2 —, —(CH 2 ) 2 —, or —(CH 2 ) 3 —.

[0313] In some embodiments, each linker in Table 7 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 7 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 7 is substituted with one water soluble polymer.

[0314] In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U. In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U via a spacer element.

[0315] In some embodiments, the water-soluble polymer is a polysaccharide.

[0316] In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0317] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0318] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0319] In some further embodiments, the phosphate-based linker of the present disclosure is a linker selected from the group of linkers listed in Table 8.

[0320] [Table 8]

[0321] In some embodiments, each n in Table 8 is independently an integer from 1 to 10. In some embodiments, each n in Table 8 is independently 1, 2, or 3.

[0322] In some embodiments, each alkylene in Table 8 is independently —(CH 2 —, —(CH 2 ) 2 —, or —(CH 2 ) 3 —.

[0323] In some embodiments, each linker in Table 8 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 8 is substituted with one or more water soluble polymers. In some embodiments, each U in Table 8 is substituted with one water soluble polymer.

[0324] In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U. In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of group U via a spacer element.

[0325] In some embodiments, the linker is * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-U-alkylene-+, During the ceremony, U is

[0326] [ka] is selected from the group consisting of Each alkylene independently represents -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -(CH2) 11 - and -(CH2) 12 - selected from the group consisting of *represents the point of attachment to the drug, for example, the —O— atom of formula (I), or formula (Ia), or formula (Ib), or formula (Ic), or formula I(d), + indicates a point of attachment to a moiety such as a reactive moiety, Each such linker is optionally substituted with one or more water-soluble polymers.

[0327] In some embodiments, the linker is substituted with one or more water-soluble polymers.

[0328] In some embodiments, the linker comprises one water-soluble polymer. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of the group U. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of the group U via a spacer element.

[0329] In some embodiments, the water-soluble polymer is a polysaccharide.

[0330] In some embodiments, the water-soluble polymer is a PEG moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0331] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) nCH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0332] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0333] In some embodiments, U is

[0334] [ka] In some embodiments, U is

[0335] [ka] In some other embodiments, U is

[0336] [ka] is.

[0337] In some embodiments, each U is conjugated to a water-soluble polymer. In some embodiments, the water-soluble polymer is conjugated to an amino acid side chain of the group U.

[0338] In some embodiments, the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element. In some embodiments, when the water-soluble polymer is conjugated to the amino acid side chain of group U via a spacer element, the spacer element is a carbonyl group.

[0339] In some embodiments, the linker has the following structure:

[0340] [ka] wherein * indicates the point of attachment to the drug, for example, the -O- atom of Formula (I), or (Ia), or (Ib), or (Ic), or Formula I(d), and + indicates the point of attachment to the reactive moiety.

[0341] In some embodiments, the linker has the following structure:

[0342] [ka] and * indicates the point of attachment to the drug, e.g., the -O- atom of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic) or Formula I(d), and + indicates the point of attachment to the reactive moiety.

[0343] In some embodiments, the linker has the following structure:

[0344] [ka] wherein T is a water-soluble polymer and R t is H or methyl, * indicates the point of attachment to the drug, for example, the -O- atom of Formula (I), or Formula (Ia), or Formula (Ib), or Formula (Ic) or Formula I(d), and + indicates the point of attachment to the reactive moiety.

[0345] In some embodiments, one or more water-soluble polymers conjugated to the linker are (polyethylene) glycol (PEG) moieties.

[0346] In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of a linker comprising group U. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of group U via a spacer element.

[0347] In some embodiments, the water-soluble polymer is a polysaccharide.

[0348] In some embodiments, the water-soluble polymer is a PEG moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0349] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0350] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0351] In some embodiments, a phosphate-based linker of the present disclosure is connected to a drug and also to a reactive moiety. Thus, the linker bridges the drug and the reactive moiety. The reactive moiety can be one that can react with another portion of a natural or unnatural amino acid of a polypeptide, such as an antibody, antibody fragment, or variant thereof, of the present disclosure, as disclosed herein.

[0352] In some other embodiments, the phosphate-based linkers disclosed herein are connected to a drug and also to an antibody, antibody fragment, or variant thereof via a linking or adduct moiety, thus bridging the drug and the antibody, antibody fragment, or variant thereof.

[0353] Drugs and Drug-Linkers In some aspects, the disclosure provides a drug or drug-linker, wherein the drug is a cytotoxic drug or agent. In some aspects of the disclosure, the cytotoxic drug is a duocarmycin. In some aspects, the cytotoxic agent is a duocarmycin analog. In some embodiments, the drug or drug-linker is a drug or drug-linker produced as described in the Examples herein, and the linker, if present, can be derivatized with a reactive or other moiety or a metabolite thereof.

[0354] In some embodiments, the compound of formula (X) has the following general structure:

[0355] [ka] (In the formula, A is an optionally substituted bicyclic ring system containing one or more nitrogen ring atoms, B is a carbonyl group, R is H or LW, L is a linker, and W is a reactive moiety; or a salt thereof. In some embodiments, the salt can be a pharmaceutically acceptable salt. In some embodiments, the bicyclic ring system A is linked to the carbonyl group B via one of the one or more nitrogen ring atoms of the bicyclic ring system A.

[0356] In some embodiments, the bicyclic ring system A can contain 9 ring atoms.

[0357] In some other embodiments, the bicyclic ring system A can contain 10 ring atoms.

[0358] In some embodiments, the bicyclic ring system A can contain 9 or 10 ring atoms, wherein the ring atoms are selected from the group consisting of carbon atoms and nitrogen atoms.

[0359] In some embodiments, the bicyclic ring system A comprises a 5-membered ring fused to a 6-membered ring, wherein the 5-membered ring comprises a nitrogen atom that links the bicyclic ring system A to the carbonyl group B. In some embodiments, the 5-membered ring is a pyrrolidine ring.

[0360] In some embodiments, the six-membered ring is an aromatic ring containing 0 or 1 nitrogen atom.

[0361] In some embodiments, bicyclic ring system A comprises a first six-membered ring fused to a second six-membered ring, the first six-membered ring comprising a nitrogen atom linking bicyclic ring system A to carbonyl group B. In some embodiments, the first six-membered ring is a piperidine ring. In some embodiments, the second six-membered ring is an aromatic ring comprising zero or one nitrogen atom.

[0362] In some embodiments, the bicyclic ring system A is hydrophobic. As will be appreciated by those skilled in the art, the hydrophobicity of a compound can be estimated from its ClogP value and calculated from its structure, with a lower ClogP value indicating a more hydrophilic molecule and a higher ClogP value indicating a more hydrophobic molecule. Thus, in some embodiments, the hydrophobicity of A is characterized by its ClogP value. In some embodiments, the hydrophobicity of A is characterized by the ClogP value of its corresponding amine "AH."

[0363] In some embodiments, AH (the corresponding amine of A) has a ClogP value of at least about 1. ClogP values ​​can be calculated using tools such as ChemDraw Professional Software (PerkinElmer Informatics). ChemDraw Professional Version 20.1.1.125 was used to calculate the ClogP values ​​reported below for some non-limiting examples of AH groups of the present disclosure.

[0364] [ka]

[0365] In some embodiments, R is H.

[0366] In some embodiments, the compound is a drug-linker compound and R is L. In some embodiments, L is a phosphate-based linker.

[0367] Thus, in some embodiments, a compound of formula (I) having the following structure:

[0368] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; A has the following structures, formulas (a), (b), (c), and (d):

[0369] [ka] is a bicyclic ring system selected from the group consisting of During the ceremony, each X 1 is C(R 1a )(R 1b ) and each R 1a and R 1bare independently H, halogen, alkyl, alkenyl, or alkynyl; each X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 3 is C, each X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(Rb ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c, -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 8 is C, each X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 or a salt thereof.

[0370] In some embodiments, AH (the corresponding amine of moiety A) has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (a) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (b) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (c) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (d) and AH has a ClogP value of at least about 1.

[0371] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0372] In some embodiments, each X 1 and X 2 is CH2 and X 9 is CH2, if present.

[0373] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0374] In some embodiments, X 4 is C(R 4 ) or N, and R 4is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0375] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0376] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0377] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0378] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X7 At least one of the groups is CH.

[0379] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0380] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0381] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0382] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0383] In some embodiments, R is H.

[0384] In some embodiments, a compound of Formula (Ia) having the following structure:

[0385] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(Ra )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 The compound of formula (I) is provided.

[0386] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1bare independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0387] In some embodiments, each X1 and X 2 is CH2 and X 9 is CH2, if present.

[0388] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0389] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0390] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R eis independently H, alkyl, alkenyl, or alkynyl.

[0391] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0392] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0393] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0394] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0395] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7is N. In some embodiments, X 4 is CH.

[0396] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0397] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0398] In some embodiments, R is H.

[0399] In some embodiments, a compound of Formula (Ib) having the following structure:

[0400] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, At this time, Each R a and R bare independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 The compound of formula (I) is provided.

[0401] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0402] In some embodiments, each X 1 and X 2 is CH2 and X 9 is CH2, if present.

[0403] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7) or N, and R 7 is H, halogen or heteroalkyl.

[0404] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0405] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0406] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0407] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0408] In some embodiments, X 4 is C(R4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0409] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0410] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0411] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0412] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0413] In some embodiments, R is H.

[0414] In some embodiments, a compound of Formula (Ic) having the following structure:

[0415] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O)m (R s ) and X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, X 9 is C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R sare independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 The compound of formula (I) is provided.

[0416] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0417] In some embodiments, each X 1 and X 2 is CH2;X 9 is CH2, if present.

[0418] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0419] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0420] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0421] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0422] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0423] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0424] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0425] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0426] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0427] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0428] In some embodiments, R is H.

[0429] In some embodiments, a compound of Formula (Id) having the structure:

[0430] [ka] (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; X1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, X 9 is C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 The compound of formula (I) is provided.

[0431] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0432] In some embodiments, each X 1 and X 2 is CH2;X 9 is CH2, if present.

[0433] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0434] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0435] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R kare independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0436] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0437] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0438] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0439] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0440] In some embodiments, X 4 is C(R 4 ) and X5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0441] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0442] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0443] In some embodiments, R is H.

[0444] In some embodiments, compounds of formula (I) are provided wherein R is H. In some embodiments, compounds of formula (Ia) are provided wherein R is H. In some embodiments, the compound is

[0445] [ka] and salts thereof. In some embodiments, the salt is a pharmaceutically acceptable salt.

[0446] In some embodiments, a compound of Formula (Ia) having the following structure:

[0447] [ka] or a salt thereof.

[0448] In some embodiments, a compound of Formula (Ia) having the following structure:

[0449] [ka] or a salt thereof.

[0450] In some embodiments, a compound of Formula (Ia) having the following structure:

[0451] [ka] or a salt thereof.

[0452] In some embodiments, a compound of Formula (Ia) having the following structure:

[0453] [ka] or a salt thereof.

[0454] In some aspects, there is provided a drug-linker compound of Formula (I) where R is LW. Thus, in some embodiments, there is provided a drug-linker compound of Formula (IL) having the following structure:

[0455] [ka] (In the formula, R is LW, where L is a linker and W is a reactive moiety; A is a compound of formula (a), (b), (c), and (d) having the following structure:

[0456] [ka] is a bicyclic ring system selected from the group consisting of During the ceremony, each X1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; each X 3 is C, each X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and each X 8 is C, each X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 or a salt thereof.

[0457] In some embodiments, AH (the corresponding amine of moiety A) has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (a) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (b) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (c) and AH has a ClogP value of at least about 1. In some embodiments, A has the structure of formula (d) and AH has a ClogP value of at least about 1.

[0458] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8 is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0459] In some embodiments, each X 1 and X 2 is CH2;X 9 is CH2, if present.

[0460] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0461] In some embodiments, X 4 is C(R 4 ) or N, and R 4is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0462] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0463] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0464] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0465] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X7 At least one of the groups is CH.

[0466] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0467] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0468] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0469] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0470] In some embodiments, L is a phosphate-based linker comprising a phosphate-based moiety selected from the group consisting of phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosporamidate, pyrophosporamidate, triphosphoramidate, tetraphosphoramidate, phosphorothioate, and diphosphorothioate.

[0471] In some embodiments, the Drug-Linker comprises a phosphate-based moiety. In some embodiments, the phosphate-based moiety is selected from the group consisting of phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosporamidate, pyrophosporamidate, triphosphoramidate, tetraphosphoramidate, phosphorthioate, and diphosphorthioate. In some embodiments, the phosphate-based moiety is a pyrophosphate ester. In some embodiments, the phosphate-based moiety is a diphosphonate.

[0472] In some embodiments, the phosphate-based linker is a bivalent linker.

[0473] In some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the -O- atom of the drug. Thus, in some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the -O- atom of a compound of Formula (X) or Formula (I), as disclosed herein, via the phosphorus atom of the phosphate-based moiety.

[0474] In some embodiments, the phosphate-based linker further comprises at least one additional moiety. In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, -(alkylene-O)-, optionally substituted arylene, -O-, -C(O)-, -N(R w )-, -S(O) 0-2-, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkylalkynyl. w is independently H or unsubstituted C1-C8 alkyl. In yet some further embodiments, each R w is independently H or methyl.

[0475] In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R w )-, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkylalkynyl. w is independently H or unsubstituted C1-C8 alkyl. In yet some further embodiments, each R w is independently H or methyl.

[0476] It is understood that each at least one additional moiety that can be present in a phosphate-based linker of the present disclosure can occur more than once within the linker. In a non-limiting example, a phosphate-based linker of the present disclosure can include one or more unsubstituted alkylene groups, and each unsubstituted alkylene group can be the same or different. In another non-limiting example, a phosphate-based linker of the present disclosure can include one or more amino acids, and each amino acid can be the same or different.

[0477] In some embodiments, L comprises at least one alkylene group.

[0478] In some embodiments, L comprises at least one amino acid. In some embodiments, L comprises one amino acid. In some embodiments, the amino acid is selected from the group consisting of serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and N. e In some embodiments, the amino acid is selected from the group consisting of lysine or N-methyl-lysine. e -methyl-lysine.

[0479] In some embodiments, L comprises one or more water-soluble polymers. In some embodiments, L comprises one water-soluble polymer.

[0480] In some embodiments, L comprises a water soluble polymer and an amino acid, and the water soluble polymer is conjugated to the amino acid. In some embodiments, the water soluble polymer is conjugated to a side chain of the amino acid. In some embodiments, the water soluble polymer is conjugated to the amino acid via a spacer element.

[0481] In some embodiments, L is selected from the group of linkers listed in Table 6.

[0482] In some embodiments, L is selected from the group of linkers listed in Table 7.

[0483] In some embodiments, L is selected from the group of linkers listed in Table 8.

[0484] In some embodiments, L is * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-J-alkylene-+, * -P(=O)(OH)-OP(=O)(OH)-(O)-(alkylene-O) n -J-alkylene-+, * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-(O-alkylene) n -J-alkylene-+, * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-J-(alkylene-O) n - alkylene - +, * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-U-alkylene-+, * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-(O-alkylene) n -U-alkylene-+, * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-(O-alkylene) n -U-alkylene-+ and * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-U-(alkylene-O) n -Alkylene-+ wherein: Each U, independently,

[0485] [ka] is selected from the group consisting of Each J is independently

[0486] [ka] and Each alkylene independently represents -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -(CH2) 11 - and -(CH2) 12- selected from the group consisting of each n is independently an integer from 1 to 100; * indicates the connection to the -O- atom of formula (IL), + indicates connection with W, Each linker, L, is optionally substituted with one or more water-soluble polymers.

[0487] In some embodiments, each n is independently an integer from 1 to 10. In some embodiments, each n is independently 1, 2, or 3.

[0488] In some embodiments, L is substituted with one or more water-soluble polymers.

[0489] In some embodiments, L comprises a group U, and one water-soluble polymer is conjugated to an amino acid side chain of the group U. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of the group U via a spacer element.

[0490] In some embodiments, L is * -P(=O)(OH)-OP(=O)(OH)-(O)-alkylene-U-alkylene-+, During the ceremony, U is

[0491] [ka] is selected from the group consisting of Each alkylene independently represents -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -(CH2) 11 - and -(CH2) 12 - selected from the group consisting of *indicates the connection to the -O- atom of formula (IL), + indicates connection with W, L is optionally substituted with one or more water-soluble polymers.

[0492] In some embodiments, L is substituted with one or more water-soluble polymers. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of the group U. In some embodiments, one water-soluble polymer is conjugated to an amino acid side chain of the group U via a spacer element. In some embodiments, the spacer element is a carbonyl group.

[0493] In some embodiments, U is

[0494] [ka] is.

[0495] In some embodiments, L has the following structure:

[0496] [ka] wherein * indicates the connection to the -O- atom of formula (IL), and + indicates the connection to W.

[0497] In some other embodiments, L has the following structure:

[0498] [ka] wherein * indicates the connection to the -O- atom of formula (IL), and + indicates the connection to W.

[0499] In some other embodiments, L has the following structure:

[0500] [ka] wherein T is a water-soluble polymer and R t is H or methyl, * indicates the connection to the -O- atom of formula (IL), and + indicates the connection to W.

[0501] In some embodiments, the water-soluble polymer is a polysaccharide.

[0502] In some embodiments, the water-soluble polymer is a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 10,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 5,000 Da. In some embodiments, the PEG moiety has a molecular weight in the range of about 100 Da to about 1,000 Da.

[0503] In some embodiments, the PEG moiety is —(CH2CH2O) n CH3, and n is an integer from 1 to 100. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3, and n is an integer from 1 to 24. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 6 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is an integer from 8 to 12. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 8. In some embodiments, the PEG moiety is -(CH2CH2O) n CH3 and n is 12.

[0504] In some embodiments, the PEG is linear. In some embodiments, the PEG is branched, hyperbranched, or dendritic.

[0505] In some embodiments, the reactive moiety W is s-N, —OH, —SH, —NH(R j ), -C(O)R q , -C(O)OR x , -C(O)CH2NH2, activated ester, -O-NH2, maleimide, tetrazine, alkyne, cyclooctyne or E)-cyclooctene, R j is H or unsubstituted alkyl, and R q is unsubstituted alkyl, and R x is H, unsubstituted alkyl or a carboxylic acid protecting group.

[0506] In some embodiments, the reactive moiety W is

[0507] [ka] -N3, -OH, -SH, -NH(R j ), -C(O)R q , -C(O)OR x , an activated ester, -O-NH2, and an optionally substituted monocyclic or polycyclic group including cyclooctyne; At this time, R j is H or unsubstituted C1-C6 alkyl, R q is unsubstituted C1-C6 alkyl, R x is H, unsubstituted C1-C6 alkyl or a carboxylic acid protecting group; R f is H or unsubstituted C1-C6 alkyl, s is 0, 1, 2, 3, 4, 5 or 6; t is 0, 1, 2, 3, 4, 5, or 6.

[0508] In some embodiments, the optionally substituted monocyclic or polycyclic group comprising cyclooctyne is

[0509] [ka] is selected from the group consisting of:

[0510] In some embodiments, W is —ONH 2 .

[0511] In some aspects, there is provided a drug-linker compound of Formula (Ia) where R is LW. Thus, in some embodiments, there is provided a drug-linker compound of Formula (Ia) having the following structure:

[0512] [ka] (In the formula, R is LW, where L is a linker and W is a reactive moiety; X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, alkyl, alkenyl, or alkynyl; X 3 is C, X 4 is C(R 4 ) or N, and R 4 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)ORc , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 5 is C(R 5 ) or N, and R 5 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 6 is C(R 6 ) or N, and R 6 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b), -C(O)SR c or -S(O) m (R s ) and X 7 is C(R 7 ) or N, and R 7 is H, halogen, -OH, -SH, -NO2, -CN, -N3, -N(R a )(R b ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R c , -C(O)OR c , -C(O)N(R a )(R b ), -C(S)R c , -C(S)OR c , -C(S)N(R a )(R b ), -C(O)SR c or -S(O) m (R s ) and X 8 is C, At this time, Each R a and R b are independently H, alkyl, alkenyl, or alkynyl; Each R c are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R s are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3 The compound of formula (I) is provided.

[0513] In some embodiments, X 1 is C(R 1a )(R 1b ) and each R 1a and R 1b are independently H, halogen, or unsubstituted alkyl; X 2 is C(R 2a )(R 2b ) and each R 2a and R 2b are independently H, halogen, or unsubstituted alkyl; X 3 is C and X 4 is C(R 4 ) or N, and R 4 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 5 is C(R 5 ) or N, and R 5 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 6 is C(R 6 ) or N, and R 6 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 7 is C(R 7 ) or N, and R 7 is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X 8is C and X 9 If present, C(R 9a )(R 9b ) and each R 9a and R 9b are independently H, halogen, or unsubstituted alkyl.

[0514] In some embodiments, each X 1 and X 2 is CH2;X 9 is CH2, if present.

[0515] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H, halogen or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H, halogen or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H, halogen or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H, halogen or heteroalkyl.

[0516] In some embodiments, X 4 is C(R 4 ) or N, and R 4 is H or heteroalkyl, and X 5 is C(R 5 ) or N, and R 5 is H or heteroalkyl, and X 6 is C(R 6 ) or N, and R 6 is H or heteroalkyl, and X 7 is C(R 7 ) or N, and R 7 is H or heteroalkyl.

[0517] In some embodiments, each heteroalkyl is alkoxy. In some embodiments, each alkoxy is independently -OR k and each R k are independently -N(R d )(R e ) or heterocyclyl, and each R d and R e is independently H, alkyl, alkenyl, or alkynyl.

[0518] In some embodiments, each heterocyclyl contains at least one nitrogen atom.

[0519] In some embodiments, X 4 is N and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 7 is CH.

[0520] In some embodiments, X 4 is C(R 4 ) and X 5 is N and X 6 is C(R 6 ) and X 7 is C(R 7 In some embodiments, X 4 and X 7 At least one of the groups is CH.

[0521] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is N and X 7 is C(R 7 In some embodiments, X 4 and X 7At least one of the groups is CH.

[0522] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is N. In some embodiments, X 4 is CH.

[0523] In some embodiments, X 4 is C(R 4 ) and X 5 is C(R 5 ) and X 6 is C(R 6 ) and X 7 is C(R 7 )

[0524] In some embodiments, X 7 is CH. In some embodiments, X 4 is CH. In some embodiments, X 4 and X 7 is CH.

[0525] In some embodiments, L is a phosphate-based linker comprising a phosphate-based moiety. In some embodiments, the phosphate-based moiety is selected from the group consisting of phosphate ester, pyrophosphate ester, triphosphate ester, tetraphosphate ester, phosphonate, diphosphonate, phosporamidate, pyrophosporamidate, triphosphoramidate, tetraphosphoramidate, phosphorothioate, and diphosphorothioate. In some embodiments, the phosphate-based moiety is a pyrophosphate ester. In some other embodiments, the phosphate-based moiety is a diphosphonate.

[0526] In some embodiments, the phosphate-based linker is a bivalent linker.

[0527] In some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the -O- atom of the drug. Thus, in some embodiments, the phosphate-based moiety of the phosphate-based linker is covalently bonded to the -O- atom of a compound of Formula (X) or Formula (I), as disclosed herein, via the phosphorus atom of the phosphate-based moiety.

[0528] In some embodiments, the phosphate-based linker further comprises at least one additional moiety. In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, substituted alkylene, -(alkylene-O)-, optionally substituted arylene, -O-, -C(O)-, -N(R w )-, -S(O) 0-2 -, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkylalkynyl. w is independently H or unsubstituted C1-C8 alkyl. In yet some further embodiments, each R w is independently H or methyl.

[0529] In some embodiments, each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R w )-, a water-soluble polymer, and an amino acid, wherein each R w is independently H or alkyl, alkenyl, or alkynyl, and combinations thereof. In some embodiments, each R w is independently H or unsubstituted C1-C8 alky...

Claims

1. Formula (I) having the following structure: 【Chemistry 1】 (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; A has the following structures: 【Chemistry 2】 is selected from the group consisting of During the ceremony, Each X １ is C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; Each X ２ is C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; Each X ３ is C, Each X ４ is C(R ４ ) or N, and R ４ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ５ is C(R ５ ) or N, and R ５ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ６ is C(R ６ ) or N, and R ６ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ７ is C(R ７ ) or N, and R ７ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ８ is C, Each X ９ If present, C(R ９ａ ) (R ９ｂ ), and each R ９ａ and R ９ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R ａ and R ｂ are independently H, alkyl, alkenyl, or alkynyl; Each R ｃ are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R ｓ are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3; or a salt thereof.

2. A has the structure of formula (a) and the compound has the structure of formula (Ia): 【Transformation 3】 (In the formula, R is H or LW, where L is a linker and W is a reactive moiety; X １ is C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; X ２ is C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; X ３ is C, X ４ is C(R ４ ) or N, and R ４ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and X ５ is C(R ５ ) or N, and R ５ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and X ６ is C(R ６ ) or N, and R ６ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and X ７ is C(R ７ ) or N, and R ７ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and X ８ is C, At this time, Each R ａ and R ｂ are independently H, alkyl, alkenyl, or alkynyl; Each R ｃ are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R ｓ are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3; 2. The compound of claim 1, wherein

3. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is independently H, halogen, or unsubstituted alkyl; X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is independently H, halogen, or unsubstituted alkyl; X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ５ But C(R ５ ) or N, and R ５ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ６ But C(R ６ ) or N, and R ６ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ７ But C(R ７ ) or N, and R ７ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ８ is C, X ９ exists, C(R ９ａ ) (R ９ｂ ), and each R ９ａ and R ９ｂ 3. The compound of claim 1 or 2, wherein is independently H, halogen, or unsubstituted alkyl.

4. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is H, X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is H, X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, X ５ But C(R ５ ) or N, and R ５ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ６ But C(R ６ ) or N, and R ６ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ７ But C(R ７ ) or N, and R ７ is H, X ８ is C, X ９ If present, CH ２ 4. The compound of claim 1, 2 or 3, wherein:

5. The compound of any one of claims 1 to 4, wherein each heteroalkyl is an alkoxy.

6. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is H, X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is H, X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, X ５ But C(R ５ ) or N, and R ５ is H, halogen or alkoxy; X ６ But C(R ６ ) or N, and R ６ is H, halogen or alkoxy; X ７ But C(R ７ ) or N, and R ７ is H, X ８ is C, X ９ If present, CH ２ The compound according to any one of claims 1 to 5,

7. X ５ But C(R ５ ) or N, and R ５ is H or alkoxy, and X ６ But C(R ６ ) or N, and R ６ The compound of claim 6, wherein is H or alkoxy.

8. X ４ is N and X ５ But C(R ５ ) and X ６ But C(R ６ ) and X ７ But C(R ７ 8. The compound according to claim 1, wherein

9. X ４ But C(R ４ ) and X ５ is N and X ６ But C(R ６ ) and X ７ But C(R ７ 8. The compound according to claim 1, wherein

10. X ４ But C(R ４ ) and X ５ But C(R ５ ) and X ６ is N and X ７ But C(R ７ 8. The compound according to claim 1, wherein

11. X ４ But C(R ４ ) and X ５ But C(R ５ ) and X ６ But C(R ６ ) and X ７ But C(R ７ 8. The compound according to claim 1, wherein

12. X ４ and X ７ The compound according to any one of claims 1 to 7 and 9 to 11, wherein at least one of is CH.

13. X ４ and X ７ The compound of any one of claims 1 to 12, wherein each of is CH.

14. R ５ and R ６ The compound of any one of claims 1 to 13, wherein at least one of is alkoxy.

15. Each said alkoxy is independently -OR ｋ and each R ｋ are independently heterocyclyl or —N(R ｄ ) (R ｅ ), wherein said heterocyclyl contains at least one nitrogen atom, and each R ｄ and R ｅ The compound of any one of claims 5 to 14, wherein is independently H, alkyl, alkenyl, or alkynyl.

16. Each of the alkoxys is -OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ CH ３ 、-OCH(CH ３ ) ２ 、-OCH ２ CH ２ N(CH ３ ) ２ 、 【Chemistry 4】 16. The compound of claim 15 selected from the group consisting of:

17. 17. The compound of any one of claims 1 to 16, wherein R is LW.

18. 18. The compound of claim 17, wherein L is a phosphate-based linker.

19. The phosphate-based linker has the following structure: 【Transformation 5】 and a phosphate-based moiety having the formula: ＊ indicates a connection to the —O— atom at position R of Formula (I) or Formula (Ia), and L further comprises at least one additional moiety, and a wavy line in the phosphate-based moiety indicates a connection to one of the at least one additional moiety, and the at least one additional moiety is selected from the group consisting of unsubstituted alkylene, substituted alkylene, -(alkylene-O)—, optionally substituted arylene, —O—, —C(O)—, —N(R ｗ ) -, -S(O) ０－２ -, a water-soluble polymer, and an amino acid; ｗ are independently H or C １ ~C ８ 20. The compound of claim 18, wherein the aryl group is aryl, ...

20. Each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R ｗ )-, a water-soluble polymer, and an amino acid; ｗ are independently H or C １ ~C ８ 20. The compound of claim 19, wherein the aryl group is aryl, ...

21. 18. The compound of any one of claims 1 to 17, wherein R is LW and L is selected from the group of linkers in Table 6.

22. 18. The compound of any one of claims 1 to 17, wherein R is LW and L is selected from the group of linkers in Table 7.

23. 18. The compound of any one of claims 1 to 17, wherein R is LW and L is selected from the group of linkers in Table 8.

24. R is LW, and L has the following structure: 【Transformation 6】 wherein ＊ The compound according to any one of claims 1 to 17, wherein represents a connection to the -O- atom at position R of formula (I) or formula (Ia), and + represents a connection to W.

25. R is LW, and L has the following structure: 【Transformation 7】 wherein T is a water-soluble polymer and R ｔ is H or methyl, ＊ The compound according to any one of claims 1 to 17, wherein represents a connection to the -O- atom at position R of formula (I) or formula (Ia), and + represents a connection to W.

26. 26. The compound of claim 25, wherein the water-soluble polymer is a (polyethylene) glycol (PEG) moiety.

27. 27. The compound of claim 26, wherein the PEG moiety has a molecular weight in the range of about 100 Da to about 100,000 Da, about 100 Da to about 10,000 Da, about 100 Da to about 5,000 Da, or about 100 Da to about 1,000 Da.

28. The PEG moiety is —(CH ２ CH ２ O) ｎ CH ３ and n is an integer from 1 to 24.

29. The PEG moiety is —(CH ２ CH ２ O) ｎ CH ３ and n is 8, 9, 10, 11 or 12.

30. R is LW and the reactive moiety W is -N ３ , -OH, -SH, -NH(R ｊ ), -C(O)R ｑ , -C(O)OR ｘ , —C(O)CH ２ NH ２ , activated ester, —O—NH ２ , maleimide, tetrazine, alkyne, cyclooctyne, or (E)-cyclooctene, R ｊ is H or unsubstituted alkyl, and R ｑ is unsubstituted alkyl, and R ｘ The compound of any one of claims 1 to 29, wherein is H, unsubstituted alkyl or a carboxylic acid protecting group.

31. The reactive moiety W is 【Transformation 8】 -N ３ , -OH, -SH, -NH(R ｊ ), -C(O)R ｑ , -C(O)OR ｘ , activated ester, —O—NH ２ and optionally substituted monocyclic or polycyclic groups containing cyclooctyne, At this time, R ｊ is H or unsubstituted C １ ~C ６ is alkyl, R ｑ is unsubstituted C １ ~C ６ is alkyl, R ｘ is H, unsubstituted C １ ~C ６ an alkyl or carboxylic acid protecting group, R ｆ is H or unsubstituted C １ ~C ６ is alkyl, s is 0, 1, 2, 3, 4, 5 or 6; 31. The compound of claim 30, wherein t is 0, 1, 2, 3, 4, 5, or 6.

32. W is -ONH ２ The compound according to any one of claims 1 to 31,

33. The compound is 【Chemistry 9】 【Chemistry 10】 3. The compound of claim 1 or 2, selected from the group consisting of: and salts thereof.

34. The following structure: 【Chemistry 11】 3. The compound according to claim 1 or 2, which is a compound having the formula:

35. The following structure: 【Chemistry 12】 3. The compound according to claim 1 or 2, which is a compound having the formula:

36. The following structure: 【Chemistry 13】 3. The compound according to claim 1 or 2, which is a compound having the formula:

37. The following structure: 【Chemistry 14】 3. The compound according to claim 1 or 2, which is a compound having the formula:

38. 17. The compound of any one of claims 1 to 16, wherein R is H.

39. The compound is 【Chemistry 15】 3. The compound of claim 1 or 2, selected from the group consisting of: and salts thereof.

40. The compound is 【Chemistry 16】 3. The compound of claim 1 or 2, selected from the group consisting of: and salts thereof.

41. Formula (II): 【Chemistry 17】 (In the formula, Ab is an antibody, and the Ab comprises one or more unnatural amino acids; L is a linker, E is the moiety that links Ab and L, d is an integer from 1 to 10, A has the following structures: [Chemistry 18] is selected from the group consisting of During the ceremony, Each X １ is C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; Each X ２ is C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; Each X ３ is C, Each X ４ is C(R ４ ) or N, and R ４ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ５ is C(R ５ ) or N, and R ５ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ６ is C(R ６ ) or N, and R ６ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ７ is C(R ７ ) or N, and R ７ is H, halogen, -OH, -SH, -NO ２ , -CN, -N ３ , -N(R ａ ) (R ｂ ), acyl, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -C(O)R ｃ , -C(O)OR ｃ , -C(O)N(R ａ ) (R ｂ ), -C(S)R ｃ , -C(S)OR ｃ , -C(S)N(R ａ ) (R ｂ ), -C(O)SR ｃ or -S(O) ｍ (R ｓ ) and Each X ８ is C, Each X ９ If present, C(R ９ａ ) (R ９ｂ ), and each R ９ａ and R ９ｂ are independently H, halogen, alkyl, alkenyl, or alkynyl; At this time, Each R ａ and R ｂ are independently H, alkyl, alkenyl, or alkynyl; Each R ｃ are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; Each R ｓ are independently H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl; each m is independently 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof.

42. A has the following structure: 【Chemistry 19】 wherein the remaining variables are as defined in claim 41.

43. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is independently H, halogen, or unsubstituted alkyl; X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is independently H, halogen, or unsubstituted alkyl; X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ５ But C(R ５ ) or N, and R ５ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ６ But C(R ６ ) or N, and R ６ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ７ But C(R ７ ) or N, and R ７ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ８ is C, X ９ exists, C(R ９ａ ) (R ９ｂ ), and each R ９ａ and R ９ｂ is independently H, halogen, or unsubstituted alkyl.

44. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is H, X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is H, X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, X ５ But C(R ５ ) or N, and R ５ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ６ But C(R ６ ) or N, and R ６ is H, halogen, alkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heterocyclylalkyl, or heteroarylalkyl; X ７ But C(R ７ ) or N, and R ７ is H, X ８ is C, X ９ If present, CH ２ 44. The ADC of claim 41, 42 or 43, wherein:

45. 45. The ADC of any one of claims 41-44, wherein each said heteroalkyl is an alkoxy.

46. X １ But C(R １ａ ) (R １ｂ ), and each R １ａ and R １ｂ is H, X ２ But C(R ２ａ ) (R ２ｂ ), and each R ２ａ and R ２ｂ is H, X ３ is C, X ４ But C(R ４ ) or N, and R ４ is H, X ５ But C(R ５ ) or N, and R ５ is H, halogen or alkoxy; X ６ But C(R ６ ) or N, and R ６ is H, halogen or alkoxy; X ７ But C(R ７ ) or N, and R ７ is H, X ８ is C, X ９ If present, CH ２ The ADC of any one of claims 41 to 45,

47. X ５ But C(R ５ ) or N, and R ５ is H or alkoxy, and X ６ But C(R ６ ) or N, and R ６ is H or alkoxy.

48. X ４ is N and X ５ But C(R ５ ) and X ６ But C(R ６ ) and X ７ But C(R ７ The ADC of any one of claims 41 to 47, wherein

49. X ４ But C(R ４ ) and X ５ is N and X ６ But C(R ６ ) and X ７ But C(R ７ The ADC of any one of claims 41 to 47, wherein

50. X ４ But C(R ４ ) and X ５ But C(R ５ ) and X ６ is N and X ７ But C(R ７ The ADC of any one of claims 41 to 47, wherein

51. X ４ But C(R ４ ) and X ５ But C(R ５ ) and X ６ But C(R ６ ) and X ７ But C(R ７ The ADC of any one of claims 41 to 47, wherein

52. X ４ and X ７ The ADC of any one of claims 41 to 51, wherein at least one of is CH.

53. X ４ and X ７ The ADC of any one of claims 41 to 47 and 49 to 52, wherein each of is CH.

54. R ５ and R ６ The ADC of any one of claims 41 to 53, wherein at least one of is alkoxy.

55. Each said alkoxy is independently -OR ｋ and Each R ｋ are independently heterocyclyl or —N(R ｄ ) (R ｅ ), wherein said heterocyclyl contains at least one nitrogen atom, and each R ｄ and R ｅ is independently H, alkyl, alkenyl, or alkynyl.

56. Each of the above alkoxys is -OCH ３ -, -OCH ２ CH ３ -, -OCH ２ CH ２ CH ３ -, -OCH(CH ３ ) ２ -, -OCH ２ CH ２ N(CH ３ ) ２ , 【Chemistry 20】 56. The ADC of claim 55, selected from the group consisting of:

57. 57. The ADC of any one of claims 41 to 56, wherein d is 1, 2, 3, or 4.

58. 58. The ADC of any one of claims 41-57, wherein L is a phosphate-based linker.

59. The phosphate-based linker has the following structure: 【Chemistry 21】 and a phosphate-based moiety having the formula: ＊ indicates a connection to the —O— atom at position L of formula (II), wherein L further comprises at least one additional moiety, and the wavy line in the phosphate-based moiety indicates a connection to one of the at least one additional moiety; Each at least one additional moiety is independently selected from the group consisting of unsubstituted alkylene, -(alkylene-O)-, -C(O)-, -N(R ｗ )-, a water-soluble polymer, and an amino acid; ｗ are independently H or C １ ~C ８ 59. The ADC of claim 58, wherein the aryl group is aryl, ... alkyl, and combinations thereof.

60. 58. The ADC of any one of claims 41-57, wherein L is selected from the group of linkers in Table 6.

61. 58. The ADC of any one of claims 41-57, wherein L is selected from the group of linkers in Table 7.

62. 58. The ADC of any one of claims 41-57, wherein L is selected from the group of linkers in Table 8.

63. L has the following structure: 【Chemistry 22】 wherein ＊ The ADC of any one of claims 41 to 57, wherein represents a connection to the -O- atom at position L in formula (II), and + represents a connection to E.

64. L has the following structure: 【Chemistry 23】 wherein T is a water-soluble polymer and R ｔ is H or methyl, ＊ The ADC of any one of claims 41 to 57, wherein represents a connection to the -O- atom at position L in formula (II), and + represents a connection to E.

65. 65. The ADC of claim 64, wherein the water-soluble polymer is a (polyethylene) glycol (PEG) moiety.

66. 66. The ADC of claim 65, wherein the PEG moiety has a molecular weight within the range of about 100 Da to about 100,000 Da, about 100 Da to about 10,000 Da, about 100 Da to about 5,000 Da, or about 100 Da to about 1,000 Da.

67. The PEG moiety is —(CH ２ CH ２ O) ｎ CH ３ and n is an integer from 1 to 24.

68. The PEG moiety is —(CH ２ CH ２ O) ｎ CH ３ and n is 8, 9, 10, 11, or 12.

69. 69. The ADC of any one of claims 41-68, wherein E comprises an amide, ester, thioester, pyrrolidine-2,5-dione, oxime, 1,2,3-triazole, or 1,4-dihydropyridazine, wherein the 1,2,3-triazole and the 1,4-dihydropyridazine are each optionally fused to an 8-membered ring.

70. E, 【Chemistry 24】 is selected from the group consisting of In the formula, each R ｊ are independently H or unsubstituted C １ ~C ６ alkyl, and each R ｑ are independently unsubstituted C １ ~C ６ alkyl, and each R ｆ are independently H or unsubstituted C １ ~C ６ 70. The ADC of claim 69, wherein: R is alkyl; each s is independently 0, 1, 2, 3, 4, 5, or 6; each t is independently 0, 1, 2, 3, 4, 5, or 6; each + indicates a connection to L; and each wavy line indicates a connection to Ab.

71. E, 【Chemistry 25】 wherein R ｑ is unsubstituted C １ ~C ６ The ADC of any one of claims 41 to 70, which is alkyl.

72. R ｑ is methyl.

73. 73. The ADC of any one of claims 41-72, wherein E links L to the unnatural amino acid of the Ab.

74. The ADC of any one of claims 41 to 73, wherein the Ab is configured to bind to an antigen.

75. The antigen is selected from the group consisting of PD-1, PD-L1, PSMA, CD70, CD3, HER2, HER3, TROP2, GPC3, VEGFR, EGFR, c-Met (HGFR), CD19, CD22, CD25 (IL-2R alpha), CD30, CD33, CD37, CD46, CD48, CD56 (NCAM-1), CD71 (transferrin R), CD74, CD79b, CD123 (IL-3R alpha), CD138 (syndecan-1), CD142, CD166 (ALCAM), CD203c (ENPP3), CD205 (LY75), CD221 (IGF-1R), CD262 (TRAIL R2), CD276 (B7-H3), mesothelin, EpCAM, CEACAM5, CEACAM6, DLL3, ROR1, ROR2, GPNMB, GCC, GUCY2c, NaPi2b, Flt-1 , Flt-3, folate receptor alpha, tissue factor (TF), CA6, MUC1, MUC16 (CA-125), BCMA, SLAMF7 (CS1), TIM1, CanAg, Ckit (CD117), E 75. The ADC of claim 74, wherein the ADC is selected from the group consisting of phA2, nectin4, SLTRK6, FGFR2, LYPD3 (C4.4a), cadherin 3, 5T4 (TPBG), STEAP1, PTK7, ephrin-A4, LIV-1 (SLC39A6 or ZIP6), SLC1A5, TENB2, ETBR, integrin v3, Crypto, AGS-5 (SLC44A4), LY6E, AXL, LAMP1, LRRC15, TNF-alpha, and MN / CA IX.

76. 76. The ADC of claim 75, wherein the antigen is TROP2, CD70, HER2, PSMA, HER3, or GPC3.

77. 77. The ADC of any one of claims 41 to 76, wherein the Ab is an anti-CD70 antibody comprising a sequence listed in Table 2.

78. 78. The ADC of claim 77, wherein the anti-CD70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:

26.

79. 79. The ADC of claim 77 or 78, wherein the anti-CD70 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO:

27.

80. 80. The ADC of claim 77, 78, or 79, wherein the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

25.

81. 80. The ADC of claim 77, 78, or 79, wherein the anti-CD70 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

20.

82. The ADC of any one of claims 77 to 81, wherein the anti-CD70 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:

19.

83. 78. The ADC of claim 77, wherein the anti-CD70 antibody comprises two heavy chains, each having the amino acid sequence of SEQ ID NO: 20, and two light chains, each having the amino acid sequence of SEQ ID NO:

19.

84. 77. The ADC of any one of claims 41 to 76, wherein the Ab is an anti-TROP2 antibody comprising a sequence listed in Table 1.

85. 85. The ADC of claim 84, wherein the anti-TROP2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

5.

86. 86. The ADC of claim 84 or 85, wherein the anti-TROP2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:

4.

87. 77. The ADC of any one of claims 41 to 76, wherein the Ab is an anti-HER2 antibody comprising a sequence listed in Table 3.

88. 88. The ADC of claim 87, wherein the anti-HER2 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

29.

89. 89. The ADC of claim 87 or 88, wherein the anti-HER2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:

30.

90. 77. The ADC of any one of claims 41 to 76, wherein the Ab is an anti-PSMA antibody comprising a sequence listed in Table 4.

91. 91. The ADC of claim 90, wherein the anti-PSMA antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

39.

92. 92. The ADC of claim 90 or 91, wherein the anti-PSMA antibody comprises a light chain having the amino acid sequence of SEQ ID NO:

40.

93. 77. The ADC of any one of claims 41 to 76, wherein the Ab is an anti-HER3 antibody comprising a sequence listed in Table 5.

94. 94. The ADC of claim 93, wherein the anti-HER3 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:

58.

95. 95. The ADC of claim 93 or 94, wherein the anti-HER3 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:

47.

96. 96. The ADC of any one of claims 41-95, wherein the antibody comprises two heavy chains and one unnatural amino acid is incorporated into each of the heavy chains.

97. 97. The ADC of any one of claims 41-96, wherein the unnatural amino acid is para-acetyl-L-phenylalanine.

98. 98. A pharmaceutical composition comprising a compound of any one of claims 1 to 40, or an ADC of any one of claims 41 to 97, and at least one pharmaceutically acceptable adjuvant, binder, buffer, carrier, diluent, or excipient.

99. 100. A method of treating a disease or condition in a subject, said method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 40, an ADC of any one of claims 41 to 97, or a pharmaceutical composition of claim 98.

100. 100. The method of claim 99, wherein the disease or condition is cancer.

101. 101. The method of claim 100, wherein the cancer is a CD70-expressing cancer.

102. 102. The method of claim 100 or 101, wherein the cancer is renal cell carcinoma.

103. 102. The method of claim 100 or 101, wherein the cancer is a hematological cancer.

104. 104. The method of claim 103, wherein the hematological cancer is leukemia, lymphoma, or myeloma.

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